Methods for enhancing stress tolerance in plants and compositions thereof

ABSTRACT

Increased tolerance to abiotic stress in a plant is provided by introducing DNA expressing a cold shock protein, e.g. bacterial cold shock protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/821,846, filed Jun. 23, 2010, now U.S. Pat. No. 8,735,657, issued May27, 2014, and incorporated herein by reference in its entirety, which isa continuation of U.S. non-provisional patent application Ser. No.10/953,856, filed Sep. 29, 2004, now U.S. Pat. No. 7,786,353, issuedAug. 31, 2010 and incorporated herein by reference it its entirety,which claims benefit under 35 USC §119(e) of U.S. provisionalapplication Ser. No. 60/506,717 filed Sep. 29, 2003 and Ser. No.60/530,453, filed Dec. 17, 2003.

INCORPORATION OF SEQUENCE LISTING

A copy of the sequence listing in a computer-readable form and named47_(—)21_(—)51768E_CSPST25.txt, which is approximately 100,395 bytes(measured in MS-Windows™) and was created on Mar. 7, 2014, is filedherewith via the USPTO EFS system and is hereby incorporated byreference.

FIELD OF THE INVENTION

This invention relates to cold, drought, salt, cold germination, heat,and other abiotic stress tolerance in plants and viral, fungal,bacterial and other abiotic stress tolerance in plants. Specificallythis invention relates to a method of increasing the biotic and abioticstress tolerance of plants by expressing a cold shock protein(s) withinthe cells of said plant.

BACKGROUND

Seed and fruit production are multi-billion dollar commercial industriesand primary sources of income for numerous states in the United Statesand for many countries around the world. Commercially valuable seedsinclude, for example, canola, cottonseeds and sunflower seeds, which areprized for the vegetable oil that can be pressed from the seed. Theseeds of leguminous plants such as peas, beans, and lentils also arecommercially valuable as they are rich in proteins, with soybeans, forexample, consisting of 40-45% protein and 18% fats and oils. Inaddition, coffee is a valuable crop made from the dried and roastedseeds of Coffea arabica plants, while chocolate is made from the cacaoseed or “bean.” Similarly, many fruits and seeds are commerciallyvaluable, including, for example, corn, rice, wheat, barley and othercereals, nuts, legumes, tomatoes, and citrus fruits. For example, cornseeds are made into many food items or items used in cooking, such astaco shells, corn oil, tortillas, corn flakes, corn meal, and manyothers. Corn is also used as raw material in many production processes,including but not limited to, feed and ethanol production.

Seed and fruit production are both limited inherently due to biotic andabiotic stress. Soybean (Glycine max), for instance, is a crop speciesthat suffers from loss of seed germination during storage and fails togerminate when soil temperatures are cool (Zhang et al., Plant Soil 188:(1997)). This is also true in corn and other plants of agronomicimportance. Improvement of abiotic stress tolerance in plants would bean agronomic advantage to growers allowing increasing growth and/orgermination in cold, drought, flood, heat, UV stress, ozone increases,acid rain, pollution, salt stress, heavy metals, mineralized soils, andother abiotic stresses. Biotic stress, such as fungal and viralinfection, also cause large crop losses world wide.

Traditional breeding (crossing specific alleles of one genotype intoanother) has been used for centuries to increase biotic stresstolerance, abiotic stress tolerance, and yield. Traditional breeding islimited inherently to the limited number of alleles present in theparental plants. This in turn limits the amount of genetic variabilitythat can be added in this manner. Molecular biology has allowed theinventors of the instant invention to look far and wide for genes thatwill improve stress tolerance in plants. Our inventors sought todetermine how other organisms react to and tolerate stressfulconditions. The cold shock proteins are part of a system used bybacteria and other organisms to survive cold and stressful conditions.It was posited by the inventors that placing genes encoding the coldshock proteins, and proteins related to them, into plants and expressingthem would increase the cold, drought, heat, water, and other abioticstress tolerance of plants as well as fungal, viral, and other bioticstress tolerance of plants. They also believe that using genes that arehomologous to cold shock proteins, or have sequence similarity, wouldalso increase biotic and abiotic stress tolerance.

This invention is useful to farmers to limit their losses due to bioticand abiotic stress.

SUMMARY OF THE INVENTION

The present invention provides a plant expressing a cold shock protein(Csp) in the cells of the plant. The expression of this csp leads togreater abiotic stress tolerance within said plant. In one embodiment, apolynucleotide encoding a csp is expressed by an operably linkedpromoter that functions in plants, and a terminator that functions inplants.

More specifically the invention provides a recombinant DNA molecule thatcomprises, in the 5′ to 3′ direction, a first DNA polynucleotide thatcomprises a promoter that functions in plants, operably linked to asecond DNA polynucleotide that encodes a cold shock protein, operablylinked to a 3′ transcription termination DNA polynucleotide providing apolyadenylation site. The first DNA polynucleotide is oftenadvantageously heterologous to the second DNA polynucleotide. Theinvention also provides a recombinant DNA molecule having an introninserted between the first DNA polynucleotide and the second DNApolynucleotide. The invention also provides a recombinant DNA moleculewhere the second DNA polynucleotide encodes a protein comprising themotif in SEQ ID NO: 3. In specific embodiments of the recombinant DNA ofthis invention the second DNA polynucleotide encodes a protein selectedfrom the group consisting of

-   -   (a) a protein with an amino acid sequence of substantial        identity to an amino acid sequence of a cold shock protein from        gram positive bacteria,    -   (b) a cold shock protein from Bacillus subtilis,    -   (c) a homologue of Bacillus subtilis cold shock protein B        (CspB),    -   (d) a protein with an amino acid sequence of substantial        identity to SEQ ID NO: 2,    -   (e) a protein with an amino acid sequence of substantial        identity to an amino acid sequence of a cold shock protein from        a gram negative bacteria,    -   (f) a protein comprising a cold shock protein from Escherichia        coli,    -   (g) a homologue of Escherichia coli cold shock protein A (CspA),    -   (h) a protein with an amino acid sequence that has substantial        identity to SEQ ID NO:1,    -   (i) a cold shock protein from Agrobacterium tumefaciens, and    -   (j) a protein having an amino acid sequence of substantial        identity to any of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21,        23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,        55, 57, 59, 61, 63, or 65.        The invention also provides a recombinant DNA molecule wherein        the promoter is selected from the group consisting of inducible        promoters, constitutive promoters, temporal-regulated promoters,        developmentally-regulated promoters, tissue-preferred promoters,        cold enhanced promoters, cold-specific promoters, stress        enhanced promoters, stress specific promoters, drought inducible        promoters, water deficit inducible promoters, and        tissue-specific promoters.

The invention also provides plant cells and plants containing in theirgenome recombinant DNA molecules as described and the propagules andprogeny produced therefrom. Plants include, but are not limited to cropplants, monocots, and dicots. More specifically these could includesoybean, corn, canola, rice, cotton, barley, oats, turf grasses, cotton,and wheat.

The invention also provides abiotic stress-tolerant, transgenic plantsthat have been transformed with a recombinant DNA molecule thatexpresses a cold shock protein. Such plants and their cells andpropagules such as seeds contain in their genome recombinant DNAmolecules that expresses a cold shock protein. Such plants exhibit oneor more of the following enhanced properties: a higher growth rate underconditions where cold temperature would be limiting for growth for anon-transformed plant of the same species,

-   -   (a) a higher growth rate under conditions where high temperature        would be limiting for growth for a non-transformed plant of the        same species,    -   (b) a higher growth rate under conditions where water would be        limiting for growth for a non-transformed plant of the same        species,    -   (c) a higher growth rate under conditions where increased salts        or ions in the soil and/or water would be limiting for growth of        a non-transformed plant of the same species,    -   (d) has a greater percentage of plants surviving after a cold        shock than a non-transformed plant of the same species,    -   (e) an increased yield when compared to a non-transformed plant        of the same species, or    -   (f) resistance to drought compared to a non-transformed plant of        the same species.

A method of the invention comprises propagating plants of thisinvention, e.g. for the purpose of generating seeds, by simply plantingsuch seeds in soil and allowing them to grow, e.g. under stressconditions. More specifically, this invention provides a method ofproducing a plant that has enhanced trait such as abiotic stresstolerance, increased yield or increased root mass. The method comprisesthe steps of

a) inserting into the genome of a plant cell or cells a recombinant DNAmolecule comprising DNA encoding a cold shock protein,

b) obtaining a transformed plant cell or cells,

c) regenerating plants from said transformed plant cell(s); and

d) selecting plants which exhibit the enhance trait.

In one aspect of the invention plants are selected which exhibitenhanced abiotic stress tolerance selected from the group consisting ofheat tolerance, salt tolerance, drought tolerance, and survival aftercold shock.

The invention also provided isolated proteins which are at least 40%identity to a protein having an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, and 65. In certain aspects comparable traits can be achieved bysubstituting a cold shock protein with a protein having higher homologythan 40% identity, e.g. with a protein that is at least 50%, 60%, 70%,80%, 90% or at least 95% identical to a cold shock protein specificallydisclosed herein. Likewise, this invention also provides an isolatednucleic acid encoding a cold shock protein motif which hybridizes to anucleic acids with a DNA sequence selected from the group comprising SEQID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 90 and 92.

The invention also specifically provides isolated nucleic acids encodinga cold shock protein which has a DNA sequence that is substantiallyidentical to a sequence in the group consisting of SEQ ID NOs: 5, 7, 9,29, 31, 33, 35, 37, 39, 41, 43, 53, 55, 57, 59, 61, 63, and 65.

The invention also provides propagules containing the above recombinantDNA molecules, when they are planted or otherwise caused to germinate,and a field of plants germinated from said propagules, e.g. where suchpropagule are seeds.

The invention also provides a method of producing seed comprisingplanting a seed of claim 59 in soil;

-   -   b) harvesting seed from said plants; and the seed produced        therefrom.

A method of producing a transgenic plant is also provided, the methodcomprising the steps of: (i) introducing into the genome of a plant cella DNA molecule comprising a DNA polynucleotide at least 40% homologousto a protein having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,and 65, or fragment, or cis element thereof, wherein said DNApolynucleotide is operably linked to a promoter and operably linked to a3′ transcription termination DNA polynucleotide; and (ii) selecting saidtransgenic plant cell; and (iii) regenerating said transgenic plant cellinto a transgenic plant; also provided are the plants made by thismethod.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plasmid map of pMON57396.

FIG. 2 shows a plasmid map of pMON23450.

FIG. 3 shows a plasmid map of pMON57397.

FIG. 4 shows a plasmid map of pMON57398.

FIG. 5 shows a plasmid map of pMON23450.

FIG. 6 shows a plasmid map of pMON57399.

FIG. 7 shows a plasmid map of pMON48421.

FIG. 8 shows a plasmid map of pMON56609.

FIG. 9 shows a plasmid map of pMON56610.

FIG. 10 shows a plasmid map of pMON73607.

FIG. 11 shows a plasmid map of pMON61322.

FIG. 12 shows a plasmid map of pMON73608.

FIG. 13 shows a plasmid map of pMON65154.

FIG. 14 shows a plasmid map of pMON72472.

FIG. 15 shows a plasmid map of pENTR1.

FIG. 16 shows the growth pattern of plants expressing the indicatedgene, and controls, showing that the genes introduced provide abioticstress tolerance.

FIG. 17 shows a plasmid map of pMON42916.

FIG. 18 shows a plasmid map of pMON73983.

FIG. 19 shows a plasmid map of pMON73984.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The instant invention provides a plant with increased tolerance tobiotic and abiotic stress. The plant provided has increased stresstolerance due to the expression of cold shock protein (csp) in the cellsof said plant. The invention provides examples of several embodimentsand contemplates other embodiments that are expected to function in theinvention.

The following definitions and methods are provided to better define thecurrent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the art. For example, definitions of common terms used in molecularbiology and molecular genetics can be found in Lewin, Genes VII, OxfordUniversity Press and Cell Press, New York, 2000; Buchanan, et al.,Biochemistry and Molecular Biology of Plants, Courier Companies, USA,2000; Lodish, et al., Molecular Cell Biology, W.H. Freeman and Co., NewYork, 2000. Common terms in genetics can be found in the priorreferences as well as Lynch, et al., Genetics and Analysis ofQuantitative Traits, Sinauer and Associates, Sunderland, Mass., 1998;Hartwell, et al., Genetics: From Genes to Genomes, McGraw-HillCompanies, Boston, Mass., 2000; Hartl, et al., Genetics: Analysis ofGenes and Genomes, Jones and Bartlett Publishers, Sudbury, Mass.;Strachan, et al., Human Molecular Genetics, John Wiley and Sons, NewYork, 1999.

The nomenclature for DNA bases as set forth in 37 CFR §1.822 is used.The standard one- and three-letter nomenclature for amino acid residuesis used.

Many agronomic traits can affect “yield”. For example, these couldinclude, without limitation, plant height, pod number, pod position onthe plant, number of internodes, incidence of pod shatter, grain size,efficiency of nodulation and nitrogen fixation, efficiency of nutrientassimilation, resistance to biotic and abiotic stress, carbonassimilation, plant architecture, resistance to lodging, percent seedgermination, seedling vigor, and juvenile traits. For example, thesecould also include, without limitation, efficiency of germination(including germination in stressed conditions), growth rate of any orall plant parts (including growth rate in stressed conditions), earnumber, seed number per ear, seed size, composition of seed (starch,oil, protein), characteristics of seed fill. Yield can be measured inmany ways, these might include test weight, seed weight, seed number perplant, seed weight per plant, seed number or weight per unit area (i.e.seeds, or weight of seeds, per acre), bushels per acre, tonnes per acre,tons per acre, kilo per hectare. In an embodiment, a plant of thepresent invention exhibits an enhanced trait that is a component ofyield.

“Nucleic acid (sequence)” or “polynucleotide (sequence)” refers tosingle- or double-stranded DNA (deoxyribonucleic acid) or RNA(ribonucleic acid) of genomic or synthetic origin, i.e., a polymer ofdeoxyribonucleotide or ribonucleotide bases, respectively, read from the5′ (upstream) end to the 3′ (downstream) end. The nucleic acid canrepresent the sense or complementary (antisense) strand.

“Native” refers to a naturally occurring (“wild-type”) nucleic acidsequence.

“Heterologous” sequence refers to a sequence which originates from aforeign source or species or, if from the same source, is modified fromits original form. For example, a native promoter could be used to causethe transcription of a heterologous gene from the same or from adifferent species.

“Parts” of a plant include all parts or pieces of a plant including, butnot limited to, roots, shoots, leaves, stems, pollen, seeds, flowers,stamen, pistils, eggs, embryos, petal, filaments, carpels (includingstigma, ovary, and style), cell(s) or any piece of the above.

“Propagule” includes all products of meiosis and mitosis, including butnot limited to, seed and parts of the plant able to propogate a newplant. For example, propagule includes a shoot, root, or other plantpart that is capable of growing into an entire plant. Propagule alsoincludes grafts where one portion of a plant is grafted to anotherportion of a different plant (even one of a different species) to createa living organism. Propagule also includes all plants and seeds producedby cloning or by bringing together meiotic products, or allowing meioticproducts to come together to form an embryo or fertilized egg (naturallyor with human intervention).

An “isolated” nucleic acid sequence is substantially separated orpurified away from other nucleic acid sequences with which the nucleicacid is normally associated in the cell of the organism in which thenucleic acid naturally occurs, i.e., other chromosomal orextrachromosomal DNA. The term embraces nucleic acids that arebiochemically purified so as to substantially remove contaminatingnucleic acids and other cellular components. The term also embracesrecombinant nucleic acids and chemically synthesized nucleic acids.

“Identity” or “identical” as used herein, when referring to comparisonsbetween protein(s) or nucleic acid(s) means 98% or greater identity.

A first nucleic acid or protein sequence displays “substantial identity”or “substantial similarity” to a reference nucleic acid sequence orprotein if, when optimally aligned (with appropriate nucleotide or aminoacid insertions or deletions totaling less than 20 percent of thereference sequence over the window of comparison) with the other nucleicacid (or its complementary strand) or protein, there is at least about60% nucleotide sequence equivalence, even better would be 70%,preferably at least about 80% equivalence, more preferably at leastabout 85% equivalence, and most preferably at least about 90%equivalence over a comparison window of at least 20 nucleotide or aminoacid positions, preferably at least 50 nucleotide or amino acidpositions, more preferably at least 100 nucleotide or amino acidpositions, and most preferably over the entire length of the firstnucleic acid or protein. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm(s),preferably by computerized implementations of these algorithms (whichcan be found in, for example, Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.).The reference nucleic acid may be a full-length molecule or a portion ofa longer molecule. Alternatively, two nucleic acids have substantialidentity if one hybridizes to the other under stringent conditions.Appropriate hybridization conditions can be determined empirically, orcan be estimated based, for example, on the relative G+C content of theprobe and the number of mismatches between the probe and targetsequence, if known. Hybridization conditions can be adjusted as desiredby varying, for example, the temperature of hybridizing or the saltconcentration (Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor Press, 1989). A first nucleic acidsequence is “operably linked” with a second nucleic acid sequence whenthe sequences are so arranged that the first nucleic acid sequenceaffects the function of the second nucleic acid sequence. Preferably,the two sequences are part of a single contiguous nucleic acid moleculeand more preferably are adjacent. For example, a promoter is operablylinked to a gene if the promoter regulates or mediates transcription ofthe gene in a cell. For example, a transcriptional termination region(terminator) is operably linked to a gene when said terminator leads toa RNA polymerase ending a transcript containing said gene at or near theterminator. For example, an enhancer is often not adjacent to thepromoter that it is exhibiting its effect on, but is generally in thesame nucleic acid molecule.

A “recombinant” nucleic acid or DNA, or RNA molecule is made by anartificial combination of two otherwise separated segments of sequence,e.g., by chemical synthesis or by the manipulation of isolated segmentsof nucleic acids by genetic engineering techniques. Techniques fornucleic-acid manipulation are well-known (see, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Press, 1989). Methods for chemical synthesis of nucleic acids arediscussed, for example, in Beaucage and Carruthers, Tetra. Letts.22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103:3185,1981. Chemical synthesis of nucleic acids can be performed, for example,on commercial automated oligonucleotide synthesizers.

“Expression” of a gene refers to the transcription of a gene to producethe corresponding mRNA and translation of this mRNA to produce thecorresponding gene product, i.e., a peptide, polypeptide, or protein.Gene expression is controlled or modulated by regulatory elementsincluding 5′ regulatory elements such as promoters.

The terms “recombinant DNA construct”, “recombinant vector”, “expressionvector” or “expression cassette” refer to any agent such as a plasmid,cosmid, virus, BAC (bacterial artificial chromosome), autonomouslyreplicating sequence, phage, or linear or circular single-stranded ordouble-stranded DNA or RNA nucleotide sequence, derived from any source,capable of genomic integration or autonomous replication, comprising aDNA molecule in which one or more DNA sequences have been linked in afunctionally operative manner.

“Complementary” refers to the natural association of nucleic acidsequences by base-pairing. Complementarity between two single-strandedmolecules may be partial, if only some of the nucleic acids pair arecomplementary; or complete, if all bases pair are complementary. Thedegree of complementarity affects the efficiency and strength ofhybridization and amplification reactions.

“Homology” refers to the level of similarity between nucleic acid oramino acid sequences in terms of nucleotide or amino acid identity orsimilarity, respectively, i.e., sequence similarity or identity.Homology, homologue, and homologous also refers to the concept ofsimilar functional properties among different nucleic acids or proteins.Homologues include genes that are orthologous and paralogous. Homologuescan be determined by using the coding sequence for a gene, disclosedherein or found in appropriate database (such as that at NCBI or others)in one or more of the following ways. For a protein sequence, thesequences should be compared using algorithms (for instance see sectionon “identity” and “substantial identity”). For nucleotide sequences thesequence of one DNA molecule can be compared to the sequence of a knownor putative homologue in much the same way. Homologues are at least 20%identical, more preferably 30%, more preferably 40%, more preferably 50%identical, more preferably 60%, more preferably 70%, more preferably80%, more preferably 88%, more preferably 92%, most preferably 95%,across any substantial (25 nucleotide or amino acid, more preferably 50nucleotide or amino acid, more preferably 100 nucleotide or amino acid,or most preferably the entire length of the shorter sequence) region ofthe molecule (DNA, RNA, or protein molecule).

Alternatively, two sequences, or DNA or RNA molecules that encode, orcan encode, amino acid sequences, are homologous, or homologues, orencode homologous sequences, if the two sequences, or the complement ofone or both sequences, hybridize to each other under stringentconditions and exhibit similar function. Thus if one were to determinewhether two protein sequences were homologues, one would both do thecomputer exercises described herein, and create degenerate codingsequences of all possible nucleic acid sequences that could encode theproteins and determine whether they could hybridize under stringentconditions. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to the high stringency ofabout 0.2×SSC at 50° C. In addition, the temperature in the wash stepcan be increased from low stringency conditions at room temperature,about 22° C., to high stringency conditions at about 65° C. Bothtemperature and salt may be varied, or either the temperature or thesalt concentration may be held constant while the other variable ischanged. In one preferred embodiment, a nucleic acid encoding a proteindescribed in the present invention will specifically hybridize to one ormore of the nucleic acid molecules or complements thereof or fragmentsof either under highly stringent conditions, for example at about2.0×SSC and about 65° C. The hybridization of the probe to the targetDNA molecule can be detected by any number of methods known to thoseskilled in the art, these, can include, but are not limited to,fluorescent tags, radioactive tags, antibody based tags, andchemiluminescent tags.

“Cold shock protein(s)” (Csp(s) or CSP(s)) are proteins that havegreater than 40% identity to Escherichia coli CspA protein (SEQ IDNO: 1) or Bacillus subtilis CspB protein (SEQ ID NO: 2), or,alternatively, cold shock proteins can be found by using the conserveddomain as determined in the literature. For example, as used herein acold shock protein is 40% identical, more preferably 50% identical, morepreferably 60% identical, more preferably 70% identical, more preferably80% identical, more preferably 90% identical, more preferably 95%identical to E. coli CspA or B. subtilis CspB across the entire lengthof E. coli CspA or B. subtilis CspB. Several databases are availablethat allow one skilled in the art to determine whether a new or existingprotein contains a cold shock domain or is a cold shock protein, fromGenbank to protein databases designed to allow the determination ofprotein relationships, and/or find related proteins. Included hereinwithin the definition are all known cold shock proteins, including butnot limited to CspA, CspB, CspC, CspD, CspE, CspF, CspG, CspH, and CspI(U.S. Pat. No. 6,610,533) from Escherichia coli.

The conserved cold shock domain is shown in SEQ ID NO: 3([FY]-G-F-I-x(6,7)-[DER]-[LIVM]-F-x-H-x-[STKR]-x-[LIVMFY]) (Prositemotif PS00352; Bucher and Bairoch, (In) ISMB-94; Proceedings 2ndInternational Conference on Intelligent Systems for Molecular Biology,Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61,AAAIPress, Menlo Park, 1994; Hofmann et al., Nucleic Acids Res. 27:215,1999). Alternatively, cold shock proteins can be found using the Sprintdatabase (a relational protein fingerprint database) (Attwood et al.,Nucleic Acids Res. 28(1):225, 2000; Attwood, et al., Nucleic AcidsResearch, 30(1), in press, 2002). Alternatively, cold shock proteins canbe found using a matrix based description, or Pfam. Pfam is a largecollection of multiple sequence alignments and hidden Markov modelscovering many common protein domains (Bateman et al., Nucleic AcidsResearch 28:263, 2000). At this writing (November 2001; Pfam release 6)there are 3071 families. Cold shock proteins are included as PF00313.The species tree showing the distribution of cold shock proteins asdetermined in the Pfam database.

“Cold shock proteins” as used herein also include, but are not limitedto, any protein that is found in a search, using a Cold shock protein asa query sequence, of a database using the “Blink” (Blast Link) functionthat can be found at the National Center for Biotechonology Information.“Blink” is a quick search function used to find proteins with similarsequences. This definition of “cold shock protein” or “cold shockdomain” is in addition to those used above, and does not replace saiddefinition. Cold shock proteins or proteins containing cold shockdomains include, but are not limited to, all currently known proteins inpublic and private databases as well as those that have yet to bediscovered that are similar enough to the claimed proteins (for example,E. coli CspA and B. subtilis CspB) to be “hits” under the standard blastsearch settings currently used in Blast Link (as of Nov. 1, 2001). As ofthis writing Blast 2 is being run, and Blast Link (“Blink”) is runningthe default parameters for protein-protein blast searches. As of thiswriting we believe the default settings used in Blink are as follows; aBLOSUM62 matrix is being run, using the “nr” database, CD search isselected, as are composition based statistics, with the complexityselected as “low complexity”, expect is 10, with a word size of 3, thegap costs are; existence 11, and extension 1. The list in Table 1 showsthe first 200 hits for E. coli CspA using these standard settings, butwe do not limit our claim to the first 200 hits. One skilled in the artwould note that under these fairly stringent criteria 167 proteins ofbacterial origin are found, but also 28 Metazoan and 5 plant proteins.These proteins include a broad range of proteins that, do to theirhomology to CspA, would be expected by the inventors to function in thepresent invention. This is by no means an all inclusive list, and otherproteins would be expected to function in the present invention.

Table 20. Some cold shock proteins and proteins containing a cold shockdomain found by similarity to E. Coli CspA. This list was compiled usingthe standard Blast Link settings at the National Center forBiotechnology information. The Genbank ID and name of each protein isshown. Note: Due to the way proteins are named, some proteins andsequences will have several entries, as proteins, cDNAs, alleles, etc.Genbank ID can be considered to be specific identifiers of each entry.Entries are in the approximate order of highest to lowest identity, incomparison with the query sequence.

Genbank ID # Gene Name 576191 Major Cold Shock Protein 7.4 (Cspa (Cs7.4)) Of (Escherichia Coli) 349561 DNA-binding protein [Salmonellatyphimurium] 3891780 Chain A, Major Cold-Shock Protein From EscherichiaColi Solution Nm 479003 cold-shock protein [Escherichia coli] 1778828major cold shock protein CSPA2 [Yersinia enterocolitica] 6073870 majorcold shock protein CSPA1 [Yersinia enterocolitica] 1468921 cold shockpotein CspG [Escherichia coli] 2275140 hypothetical protein [Yersiniapestis] 12514257 homolog of Salmonella cold shock protein [Escherichiacoli O157:H7 15981565 major cold shock protein Cspa1 [Yersinia pestis]3249024 cold shock protein CspB [Yersinia enterocolitica] 15979692 coldshock protein [Yersinia pestis] 1742550 Cold shock-like protein CspB.[Escherichia coli] 16419141 RNA chaperone, negative regulator of cspAtranscription [Salmonella 10039151 cold shock-like protein cspE[Buchnera sp. APS] 9957540 cold shock protein B [Yersiniaenterocolitica] 1778540 cold shock-like protein [Escherichia coli]471099 CspE (MsmC) [Escherichia coli] 2961317 cspB [Salmonellatyphimurium] 16503235 cold shock protein [Salmonella enterica subsp.enterica serovar 9658370 cold shock domain family protein [Vibriocholerae] 460698 CspC (MsmB) [Escherichia coli] 15980582 putative coldshock protein [Yersinia pestis] 10038996 cold shock-like protein cspC[Buchnera sp. APS] 15979774 cold shock protein [Yersinia pestis] 9657556cold shock transcriptional regulator CspA [Vibrio cholerae] 4454361 coldshock protein, CSPA [Vibrio cholerae] 2970685 cold shock protein C[Salmonella typhimurium] 1402743 major cold-shock protein [Citrobacterfreundii] 5869509 CspG [Shewanella violacea] 5869504 CspA [Shewanellaviolacea] 9968446 cold shock protein [Lactobacillus plantarum] 1405474CspC protein [Bacillus cereus] 3850776 cold shock protein D [Lactococcuslactis] 10176234 cold-shock protein [Bacillus halodurans] 1869948 coldshock protein [Lactobacillus plantarum] 729220 COLD SHOCK PROTEIN CSPC7379745 putative transcriptional regulator [Neisseria meningitidisZ2491] 1620431 csp [Lactobacillus plantarum] 1405472 CspB protein[Bacillus cereus] 3892590 cold shock protein E [Lactococcus lactis]7226073 cold-shock domain family protein [Neisseria meningitidis MC58]2493766 COLD SHOCK-LIKE PROTEIN CSPLA (CSPL) 1001878 CspA protein[Listeria monocytogenes] 13623066 putative cold shock protein[Streptococcus pyogenes M1 GAS] 758663 cold shock protein [Arthrobacterglobiformis] 4468119 cold shock protein A; CspA protein [Bordetellapertussis] 2370256 cold shock protein [Lactococcus lactis] 1405470 CspAprotein [Bacillus cereus] 2226349 CspC [Staphylococcus aureus] 1405476CspD protein [Bacillus cereus] 1513079 cold acclimation protein A[Pseudomonas fragi] 7242722 cold shock protein [Streptomyces coelicolorA3(2)] 2425105 major cold-shock protein [Micrococcus luteus] 2105046cspA [Mycobacterium tuberculosis H37Rv] 15023696 Cold shock protein[Clostridium acetobutylicum] 12720931 MsmB [Pasteurella multocida]8101860 major cold shock protein CspA [Staphylococcus aureus] 1513081cold acclimation protein B [Pseudomonas fragi] 3097243 small cold-shockprotein [Mycobacterium leprae] 9587215 cold-shock protein CspA[Mycobacterium smegmatis] 9107526 cold shock protein [Xylella fastidiosa9a5c] 1256629 cold-shock protein [Bacillus subtilis] 12054789 cold shockprotein (CspLB) [Listeria monocytogenes] 1864167 major cold-shockprotein homolog CspB [Listeria monocytogenes] 1421212 Major Cold ShockProtein (Cspb) 297761 cold shock protein (CspB) [Bacillus subtilis]13625473 cold acclimation protein CapB [Pseudomonas sp. 30/3] 9657576cold shock DNA-binding domain protein [Vibrio cholerae] 11933043cold-shock like protein [Streptomyces nodosus] 11933034 cold-shock likeprotein [Streptomyces hygroscopicus] 8248794 cold shock protein[Streptomyces coelicolor A3(2)] 1778825 major cold shock protein CspA[Pseudomonas aeruginosa] 740006 cold shock protein 2226347 CspB[Staphylococcus aureus] 1616777 cold shock-like protein [Stigmatellaaurantiaca] 7210998 cold-shock protein [Streptomyces coelicolor A3(2)]729217 COLD SHOCK PROTEIN CSPB 1067201 cold shock protein [Streptomycescoelicolor] 7321274 cold shock protein [Streptomyces coelicolor A3(2)]1402789 major cold-shock protein [Yersinia enterocolitica] 1513086temperature acclimation protein B [Pseudomonas fragi] 16411332 similarto cold shock protein [Listeria monocytogenes] 5732895 F40 [Streptomycescoelicolor A3(2)] 4193390 CspA [Myxococcus xanthus] 4193394 CspC[Myxococcus xanthus] 1405478 CspE protein [Bacillus cereus] 1402753major cold-shock protein [Klebsiella pneumoniae] 2983729 cold shockprotein [Aquifex aeolicus] 2815334 cold-shock domain protein[Streptomyces coelicolor A3(2)] 4193398 CspE [Myxococcus xanthus]4193396 CspD [Myxococcus xanthus] 2894098 cold shock protein [Thermotogamaritima] 15074838 PUTATIVE COLD SHOCK-LIKE TRANSCRIPTION REGULATORPROTEIN 1402731 major cold-shock protein [Aeromonas hydrophila] 46789 7kDa cold shock like protein [Streptomyces clavuligerus] 9946316 probablecold-shock protein [Pseudomonas aeruginosa] 1402769 major cold-shockprotein [Proteus vulgaris] 456240 major cold shock protein (CspB)[Sporosarcina globispora] 19743 nsGRP-2 [Nicotiana sylvestris] 15026046Cold shock protein [Clostridium acetobutylicum] 11493820 cold shockprotein C [Yersinia enterocolitica] 4982460 cold shock protein[Thermotoga maritima] 15979415 cold shock-like protein [Yersinia pestis]16419455 similar to CspA but not cold shock induced [Salmonellatyphimurium 14523127 putative cold shock protein [Sinorhizobiummeliloti] 9107847 temperature acclimation protein B [Xylella fastidiosa9a5c] 3036806 glycine-rich protein [Arabidopsis thaliana] 2182333 Y4cH[Rhizobium sp. NGR234] 1402733 major cold-shock protein [Aeromonassalmonicida] 9655615 cold shock-like protein CspD [Vibrio cholerae]3831556 major cold shock protein [Enterococcus faecalis] 3821915 majorcold shock protein [Lactococcus lactis subsp. cremoris] 15160284AGR_L_3376p [Agrobacterium tumefaciens] 6458627 cold shock protein, CSDfamily [Deinococcus radiodurans] 3821923 major cold shock protein[Lactobacillus helveticus] 3821911 major cold shock protein [Lactococcuslactis subsp. lactis] 15157349 AGR_C_4003p [Agrobacterium tumefaciens]15154976 AGR_C_161p [Agrobacterium tumefaciens] 3831558 major cold shockprotein [Pediococcus pentosaceus] 456238 cold shock protein [Bacillussubtilis] 117574 COLD SHOCK-LIKE PROTEIN CSPD (CSP-D) 12620649 ID534[Bradyrhizobium japonicum] 13424521 cold-shock domain family protein[Caulobacter crescentus] 3776223 CspA [Sinorhizobium meliloti] 15075353PUTATIVE COLD SHOCK TRANSCRIPTION REGULATOR PROTEIN [Sinorhizobium15075133 PROBABLE COLD SHOCK TRANSCRIPTION REGULATOR PROTEIN[Sinorhizobium 3821913 major cold shock protein [Lactococcus lactissubsp. lactis] 13476765 cold shock protein [Mesorhizobium loti] 3821925major cold shock protein [Streptococcus thermophilus] 3821921 major coldshock protein [Lactobacillus acidophilus] 729222 COLD SHOCK-LIKE PROTEINCSPJ 15162334 AGR_pAT_762p [Agrobacterium tumefaciens] 13475232 coldshock protein [Mesorhizobium loti] 9947082 probable cold-shock protein[Pseudomonas aeruginosa] 13424199 cold-shock domain family protein[Caulobacter crescentus] 9948689 cold-shock protein CspD [Pseudomonasaeruginosa] 4193392 CspB [Myxococcus xanthus] 13488430 cold shockprotein [Mesorhizobium loti] 12720739 CspD [Pasteurella multocida]3831560 major cold shock protein [Bifidobacterium animalis] 1513084temperature acclimation protein A [Pseudomonas fragi] 1169113 COLDSHOCK-LIKE PROTEIN CSPD 5714745 cold shock protein 7.4 [Rhodococcus sp.7/1] 1402767 major cold-shock protein [Photobacterium phosphoreum]14523160 probable CspA5 cold shock protein transcriptional regulator15979447 cold shock-like protein [Yersinia pestis] 13488214 cold-shockprotein [Mesorhizobium loti] 5714743 cold shock protein A [Rhodococcussp. 5/14] 3861208 COLD SHOCK-LIKE PROTEIN (cspA) [Rickettsia prowazekii]81624 glycine-rich protein 2 - Arabidopsis thaliana 15156913 AGR_C_3315p[Agrobacterium tumefaciens] 15074652 PUTATIVE COLD SHOCK TRANSCRIPTIONREGULATOR PROTEIN [Sinorhizobium 7295442 CG17334 gene product[Drosophila melanogaster] 3850772 cold shock protein A [Lactococcuslactis] 14334920 putative glycine-rich zinc-finger DNA-binding protein[Arabidopsis 3892588 cold shock protein C [Lactococcus lactis] 2708747putative glycine-rich, zinc-finger DNA-binding protein [Arabidopsis2739396 Y-box protein [Drosophila melanogaster] 1402763 major cold-shockprotein [Photobacterium mondopomensis] 15620137 cold shock-like protein[Rickettsia conorii] 1402755 major cold-shock protein [Lactobacilluscasei] 409419 Y-Box factor [Aplysia californica] 14039811 Y-box bindingprotein [Schistosoma japonicum] 9946868 probable cold-shock protein[Pseudomonas aeruginosa] 1483311 Y-box protein [Dugesia japonica]1477478 Y-box binding protein [Schistosoma mansoni] 1402759 majorcold-shock protein [Listeria innocua] 15159048 AGR_L_1288p[Agrobacterium tumefaciens] 2228815 major cold-shock protein CspH[Salmonella typhimurium] 6911694 cold-shock protein A [Streptococcusthermophilus] 2970679 Y box protein [Drosophila silvestris] 14602477Similar to cold shock domain protein A [Homo sapiens] 10727970 yps geneproduct [Drosophila melanogaster] 1402757 major cold-shock protein[Listeria grayi] 1402751 major cold-shock protein [Enterococcusfaecalis] 1083796 RYB-a protein - rat 505133 RYB-a [Rattus norvegicus]14523481 probable CspA6 cold shock protein transcriptional regulator8100512 Y-box protein ZONAB-B [Canis familiaris] 8100510 Y-box proteinZONAB-A [Canis familiaris] 15306095 hypothetical protein XP_053028 [Homosapiens] 10185725 Y-box protein 3 short isoform [Mus musculus] 10185723Y-box protein 3 long isoform [Mus musculus] 7385223 RNA binding proteinMSY4 [Mus musculus] 6166110 DNA-BINDING PROTEIN A (COLD SHOCK DOMAINPROTEIN A) 1402783 major cold-shock protein [Streptococcus pyogenes]1167838 DNA-binding protein [Homo sapiens] 1160331 dbpA murine homologue[Mus musculus] 1101884 YB2 [Rattus norvegicus] 950340 DNA-bindingprotein A [Homo sapiens] 532211 Y-box binding protein [Mus musculus]87332 DNA-binding protein A - human (fragment) 14742409 hypotheticalprotein XP_046353 [Homo sapiens] 14270385 cold-shock domain protein[Takifugu rubripes] 9653686 TSH receptor suppressor element-bindingprotein-1; TSEP-1 8249978 cold shock protein B [Streptomyces coelicolorA3(2)] 3695368 zfY1 [Danio rerio]

Bacillus subtilis (B. subtilis) CspB is a protein that accumulates inresponse to cold shock (Willimsky, et al. Journal of Bacteriology174:6326 (1992)). It has homology to CspA from E. coli (see Table I) andcontains a single stranded nucleic acid binding domain (Lopez, et al.,The Journal of Biological Chemistry 276:15511 (2001)). Using the samebasic Blast search at NCBI (Blink) the following proteins are designatedas “hits”. The number of hits shown here is limited to 200, but manyother proteins would be expected function in the invention.

TABLE 21 Some cold shock proteins and proteins containing cold shockdomains found searching with B. subtilis CspB. This list was compiledusing the standard Blast Link (Blink) settings at the National Centerfor Biotechnology Information. The Genbank ID and name of each proteinis shown. Note: Due to the way proteins are named, some proteins andsequences will have several entries, as proteins, cDNAs, alleles, etc.Genbank ID can be considered to be specific identifiers of each entry.Entries are in the approximate order of highest to lowest identity tothe query sequence. GenBank ID # Gene Name 1421212 Major Cold ShockProtein (Cspb) 1405476 CspD protein [Bacillus cereus] 729217 COLD SHOCKPROTEIN CSPB 456240 major cold shock protein (CspB) [Sporosarcinaglobispora] 1256629 cold-shock protein [Bacillus subtilis] 740006 coldshock protein 456238 cold shock protein [Bacillus subtilis] 12054789cold shock protein (CspLB) [Listeria monocytogenes] 1864167 majorcold-shock protein homolog CspB [Listeria monocytogenes] 1405472 CspBprotein [Bacillus cereus] 8101860 major cold shock protein CspA[Staphylococcus aureus] 16411332 similar to cold shock protein [Listeriamonocytogenes] 10176234 cold-shock protein [Bacillus halodurans] 2493766COLD SHOCK-LIKE PROTEIN CSPLA (CSPL) 1001878 CspA protein [Listeriamonocytogenes] 1405470 CspA protein [Bacillus cereus] 1405474 CspCprotein [Bacillus cereus] 13623066 putative cold shock protein[Streptococcus pyogenes M1 GAS] 729220 COLD SHOCK PROTEIN CSPC 2226349CspC [Staphylococcus aureus] 9968446 cold shock protein [Lactobacillusplantarum] 1402739 major cold-shock protein [Bacillus subtilis] 3892590cold shock protein E [Lactococcus lactis] 2226347 CspB [Staphylococcusaureus] 3850776 cold shock protein D [Lactococcus lactis] 1402741 majorcold-shock protein [Bacillus subtilis] 15979774 cold shock protein[Yersinia pestis] 10039151 cold shock-like protein cspE [Buchnera sp.APS] 8248794 cold shock protein [Streptomyces coelicolor A3(2)] 460698CspC (MsmB) [Escherichia coli] 11933043 cold-shock like protein[Streptomyces nodosus] 11933034 cold-shock like protein [Streptomyceshygroscopicus] 1620431 csp [Lactobacillus plantarum] 16419141 RNAchaperone, negative regulator of cspA transcription [Salmonellatyphimurium LT2] 15979692 cold shock protein [Yersinia pestis] 2894098cold shock protein [Thermotoga maritima] 1869948 cold shock protein[Lactobacillus plantarum] 2370256 cold shock protein [Lactococcuslactis] 2970685 cold shock protein C [Salmonella typhimurium] 1778540cold shock-like protein [Escherichia coli] 471099 CspE (MsmC)[Escherichia coli] 10038996 cold shock-like protein cspC [Buchnera sp.APS] 7242722 cold shock protein [Streptomyces coelicolor A3(2)] 15026046Cold shock protein [Clostridium acetobutylicum] 15980582 putative coldshock protein [Yersinia pestis] 9657576 cold shock DNA-binding domainprotein [Vibrio cholerae] 349561 DNA-binding protein [Salmonellatyphimurium] 4982460 cold shock protein [Thermotoga maritima] 1405478CspE protein [Bacillus cereus] 9946316 probable cold-shock protein[Pseudomonas aeruginosa] 9658370 cold shock domain family protein[Vibrio cholerae] 5869509 CspG [Shewanella violacea] 1067201 cold shockprotein [Streptomyces coelicolor] 9948689 cold-shockprotein CspD[Pseudomonas aeruginosa] 3891780 Chain A, Major Cold-Shock Protein FromEscherichia Coli Solution Nmr Structure 576191 Major Cold Shock Protein7.4 (Cspa (Cs 7.4)) Of (Escherichia Coli) 72232 major cold shock proteincspA - Escherichia coli 9657556 cold shock transcriptional regulatorCspA [Vibrio cholerae] 6458627 cold shock protein, CSD family[Deinococcus radiodurans] 3831556 major cold shock protein [Enterococcusfaecalis] 15023696 Cold shock protein [Clostridium acetobutylicum]2425105 major cold-shock protein [Micrococcus luteus] 1402737 majorcold-shock protein [Bacillus cereus] 9587215 cold-shock protein CspA[Mycobacterium smegmatis] 7226073 cold-shock domain family protein[Neisseria meningitidis MC58] 4454361 cold shock protein, CSPA [Vibriocholerae] 479003 cold-shock protein [Escherichia coli] 3097243 smallcold-shock protein [Mycobacterium leprae] 1778828 major cold shockprotein CSPA2 [Yersinia enterocolitica] 758663 cold shock protein[Arthrobacter globiformis] 2105046 cspA [Mycobacterium tuberculosisH37Rv] 7379745 putative transcriptional regulator [Neisseriameningitidis Z2491] 3249024 cold shock protein CspB [Yersiniaenterocolitica] 7210998 cold-shock protein [Streptomyces coelicolorA3(2)] 1513081 cold acclimation protein B [Pseudomonas fragi] 5869504CspA [Shewanella violacea] 1778825 major cold shock protein CspA[Pseudomonas aeruginosa] 1513086 temperature acclimation protein B[Pseudomonas fragi] 12514257 homolog of Salmonella cold shock protein[Escherichia coli O157:H7 EDL933] 5732895 F40 [Streptomyces coelicolorA3(2)] 3831558 major cold shock protein [Pediococcus pentosaceus]1468921 cold shock potein CspG [Escherichia coli] 13625473 coldacclimation protein CapB [Pseudomonas sp. 30/3] 6073870 major cold shockprotein CSPA1 [Yersinia enterocolitica] 1402771 major cold-shock protein[Staphylococcus aureus] 1402761 major cold-shock protein [Lactococcuslactis subsp. cremoris] 15981565 major cold shock protein Cspa1[Yersinia pestis] 9107847 temperature acclimation protein B [Xylellafastidiosa 9a5c] 7321274 cold shock protein [Streptomyces coelicolorA3(2)] 2815334 cold-shock domain protein [Streptomyces coelicolor A3(2)]2275140 hypothetical protein [Yersinia pestis] 9947082 probablecold-shock protein [Pseudomonas aeruginosa] 2983729 cold shock protein[Aquifex aeolicus] 2961317 cspB [Salmonella typhimurium] 46789 7 kDacold shock like protein [Streptomyces clavuligerus] 9107526 cold shockprotein [Xylella fastidiosa 9a5c] 1513079 cold acclimation protein A[Pseudomonas fragi] 4193394 CspC [Myxococcus xanthus] 4193392 CspB[Myxococcus xanthus] 3821911 major cold shock protein [Lactococcuslactis subsp. lactis] 16503235 cold shock protein [Salmonella entericasubsp. enterica serovar Typhi] 9957540 cold shock protein B [Yersiniaenterocolitica] 3821921 major cold shock protein [Lactobacillusacidophilus] 1616777 cold shock-like protein [Stigmatella aurantiaca]1402759 major cold-shock protein [Listeria innocua] 4468119 cold shockprotein A; CspA protein [Bordetella pertussis] 1742550 Cold shock-likeprotein CspB. [Escherichia coli] 12720739 CspD [Pasteurella multocida]3821915 major cold shock protein [Lactococcus lactis subsp. cremoris]1402765 major cold-shock protein [Pediococcus pentosaceus] 1513084temperature acclimation protein A [Pseudomonas fragi] 4193396 CspD[Myxococcus xanthus] 4193398 CspE [Myxococcus xanthus] 3831560 majorcold shock protein [Bifidobacterium animalis] 4193390 CspA [Myxococcusxanthus] 3821923 major cold shock protein [Lactobacillus helveticus]12720931 MsmB [Pasteurella multocida] 3850772 cold shock protein A[Lactococcus lactis] 9655615 cold shock-like protein CspD [Vibriocholerae] 9946868 probable cold-shock protein [Pseudomonas aeruginosa]1402757 major cold-shock protein [Listeria grayi] 3821913 major coldshock protein [Lactococcus lactis subsp. lactis] 1402735 majorcold-shock protein [Bacillus atrophaeus] 1402751 major cold-shockprotein [Enterococcus faecalis] 3892588 cold shock protein C[Lactococcus lactis] 1169113 COLD SHOCK-LIKE PROTEIN CSPD 15979415 coldshock-like protein [Yersinia pestis] 117574 COLD SHOCK-LIKE PROTEIN CSPD(CSP-D) 15075133 PROBABLE COLD SHOCK TRANSCRIPTION REGULATOR PROTEIN[Sinorhizobium meliloti] 16419455 similar to CspA but not cold shockinduced [Salmonella typhimurium LT2] 11493820 cold shock protein C[Yersinia enterocolitica] 1402783 major cold-shock protein[Streptococcus pyogenes] 3821925 major cold shock protein [Streptococcusthermophilus] 1402775 major cold-shock protein [Streptococcusdysgalactiae] 8249978 cold shock protein B [Streptomyces coelicolorA3(2)] 15160284 AGR_L_3376p [Agrobacterium tumefaciens] 81624glycine-rich protein 2 - Arabidopsis thaliana 19743 nsGRP-2 [Nicotianasylvestris] 2916930 cspB [Mycobacterium tuberculosis H37Rv] 13475232cold shock protein [Mesorhizobium loti] 3861208 COLD SHOCK-LIKE PROTEIN(cspA) [Rickettsia prowazekii] 2182333 Y4cH [Rhizobium sp. NGR234]13476765 cold shock protein [Mesorhizobium loti] 3776223 CspA[Sinorhizobium meliloti] 1402755 major cold-shock protein [Lactobacilluscasei] 15620137 cold shock-like protein [Rickettsia conorii] 15154976AGR_C_161p [Agrobacterium tumefaciens] 15074838 PUTATIVE COLD SHOCK-LIKETRANSCRIPTION REGULATOR PROTEIN [Sinorhizobium meliloti] 14548150RNA-binding cold-shock protein [uncultured crenarchaeote 4B7] 2440094small cold-shock protein [Mycobacterium leprae] 14523127 putative coldshock protein [Sinorhizobium meliloti] 12620649 ID534 [Bradyrhizobiumjaponicum] 1063684 AtGRP2b [Arabidopsis thaliana] 13424521 cold-shockdomain family protein [Caulobacter crescentus] 3036806 glycine-richprotein [Arabidopsis thaliana] 1402731 major cold-shock protein[Aeromonas hydrophila] 214642 p54 [Xenopus laevis] 15075353 PUTATIVECOLD SHOCK TRANSCRIPTION REGULATOR PROTEIN [Sinorhizobium meliloti]13424199 cold-shock domain family protein [Caulobacter crescentus]14602477 Similar to cold shock domain protein A [Homo sapiens] 1175535CYTOPLASMIC RNA-BINDING PROTEIN P56 (Y BOX BINDING PROTEIN-2) (Y-BOXTRANSCRIPTION FACTOR) (MRNP4) 104266 Y box-binding protein 2 - Africanclawed frog 15157349 AGR_C_4003p [Agrobacterium tumefaciens] 8100512Y-box protein ZONAB-B [Canis familiaris] 8100510 Y-box protein ZONAB-A[Canis familiaris] 1483311 Y-box protein [Dugesia japonica] 1402767major cold-shock protein [Photobacterium phosphoreum] 1402733 majorcold-shock protein [Aeromonas salmonicida] 15306095 hypothetical proteinXP_053028 [Homo sapiens] 14742409 hypothetical protein XP_046353 [Homosapiens] 14270385 cold-shock domain protein [Takifugu rubripes] 10185725Y-box protein 3 short isoform [Mus musculus] 10185723 Y-box protein 3long isoform [Mus musculus] 9653686 TSH receptor suppressorelement-binding protein-1; TSEP-1 [Rattus sp.] 7385223 RNA bindingprotein MSY4 [Mus musculus] 6166110 DNA-BINDING PROTEIN A (COLD SHOCKDOMAIN PROTEIN A) (SINGLE-STRAND DNA BINDING PROTEIN NF-GMB) 3695368zfY1 [Danio rerio] 2745892 Y box transcription factor [Mus musculus]2073109 Y box protein 1 [Carassius auratus] 1353778 Y-Box bindingprotein [Columba livia] 1167838 DNA-binding protein [Homo sapiens]1160331 dbpA murine homologue [Mus musculus] 1101884 YB2 [Rattusnorvegicus] 1083796 RYB-a protein - rat 988283 mYB-1b [Mus musculus]988281 mYB-1a [Mus musculus] 950340 DNA-binding protein A [Homo sapiens]608518 p50 [Oryctolagus cuniculus] 532211 Y-box binding protein [Musmusculus] 516701 similar to dbpB/YB-1 of mouse [Gallus gallus] 505133RYB-a [Rattus norvegicus] 457262 nuclease sensitive element bindingprotein-1 [Homo sapiens] 423015 nuclease sensitive element-bindingprotein 1 - human 289797 YB-1 protein [Gallus gallus] 203398 putative[Rattus norvegicus] 199821 Y box transcription factor [Mus musculus]189299 DNA-binding protein [Homo sapiens] 162983 transcription factorEF1(A) [Bos taurus] 115848 Y BOX BINDING PROTEIN-1 (Y-BOX TRANSCRIPTIONFACTOR) (YB-1) (CCAAT-BINDING TRANSCRIPTION FACTOR I SUBUNIT A) (CBF-A)(ENHANCER FACTOR I SUBUNIT A) (EFI-A) (DNA-BINDING PROTEIN B) (DBPB)112410 Y box-binding protein 1 - rat

CSPs are a group of proteins that may or may not be increased in amountwhen the temperature is lowered or other stress is applied. In fact, inthe best studied organism with respect to the cold shock proteins, E.coli, some cold shock proteins are constitutively expressed while othersare induced by cold, still others seem to be specific for specificstresses and/or growth conditions or stages. A review of this isYamanaka, et al., Molecular Microbiology, 27:247 (1998). In this reviewYamanaka and colleagues detail how the nine cold shock proteins in E.coli (CspA through CspI) are expressed. CspA, CspB, and CspG are coldinducible. CspD is induced at the stationary phase of the cell cycle andduring starvation. CspC and E have been implicated in cell division.

CspA is the major cold shock protein from Escherichia coli (E. coli)(SEQ ID NO:1). CspA is also called Major Cold Shock Protein 7.4. CspA ishighly induced in response to cold shock (Goldstein, et al., Proceedingsof the National Academy of Science (USA) 87:283 (1990)). In someconditions of slower growth, ribosomes are slowed due to RNA or DNAsecondary structure formation, and this may act as a signal for theincreased synthesis of CSPs in their native organism. CSPs bind to ssDNAand RNA under in-vitro conditions (Phadtare, et al., MolecularMicrobiology 33:1004 (1999)). CSPs are thought to bind to RNA in arelatively non-specific manner during translation and prevent secondarystructure formation and stabilize the RNA (this function is sometimesreferred as an RNA chaperone). The ribosome can then easily displace theCSPs and initiate translation on a linear RNA template. We believe thatthe present invention might involve the single stranded nucleic acidbinding function of these proteins, and this function can come from anycold shock protein or protein containing a cold shock domain, whichincludes, for example, prokaryotic cold shock proteins, eukaryotic Y-Boxcontaining genes, some glycine rich proteins (GRP), and other proteinscontaining the cold shock domain. These proteins include, but are notlimited to, those shown in FIG. 4, Trends in Biochemical Science,23(8):289 (1998) (paper included, herein incorporated by reference).This figure clearly shows the evolutionary relationship between theseproteins. The origin of these proteins likely precedes the divergence ofmodern day bacteria and eukaryotes, and it has been postulated thatthese proteins may have been present at the advent of single cellevolution, 3.5 billion years ago. We have selected two proteins totransform into plants as examples, as shown in the figure cited abovethese proteins are more greatly divergent from each other than from manyof their eukaryotic counterparts. We expect that the ectopic expressionof these proteins may improve tolerance to biotic and abiotic stresseswhich could include but are not limited to the growth, vigor, yield, andhealth of plants under a variety of stressful conditions that mayinclude cold, drought, salt stress, heat, survival after cold shock,fungal infection, viral infection, microbial infection, and coldgermination.

Another possible explanation for the increased growth rate of plantsunder stress could be the elicitation of pathogen-associated molecularpatterns (PAMP) provided by the expression of CSPs. In this model aplant would develop a PAMP response that would elicit a plant responsesomewhat like systemic acquired resistance (SAR) (much like SAR worksfor biotic stresses) as the plant would be “prepared” for the stressprior to its application. For this model to work the plant must besignaled that the CSP is present, this mechanism may have recently beenprovided through a plant receptor that binds CSP (Felix, et al, Journalof Biological Chemistry 278(8):6201-8 (2003)). This mechanism would meanthat any gene that bound a receptor which elicited a PAMP-type responsewould function in the invention. Elicitation of PAMP-type responses hasgenerally been studied for biotic stresses, and has often been elicitedthrough exogenous administration of agents. Herein we could be elicitingthe PAMP-type response to the CSP produced from the CSP transgene. Thetransgene transformed into a plant cell as part of a recombinant DNAconstruct, through a particle gun or agrobacterium mediatedtransformation. This in turn could be creating a systemic acquiredresistance type response in the plant, in turn increasing resistance toabiotic stress. This response could work in both monocots and dicots,including but not limited to corn, soybean, wheat, rice, Arabidopsis,canola, and cotton. If the above PAMP method is the mode of action forthe CSPs, then the CSP might be expected to provide biotic stressprotection as well as abiotic stress protection. None of thesemechanisms are meant to be limiting and one or both, or myriad others,could be involved in the phenotype manifested.

MF2, a Csp-like protein from Bacillus thuringensis, has been purportedto give some protection against viral infection in a plant. U.S. Pat.No. 6,528,480 shows this tolerance to biotic stress via rubbing theleaves of a plant with an extract containing the protein and infectingthe plant with a virus. They contemplate, but do not create, transgenicplants therein.

“Non-transformed plant of the same species” is meant to be inclusive ofall plants of the same species as a transformed plant. In one embodimentthe transformed plants is of the same species and strain as thetransformed plant. In another embodiment the plant is as identical aspossible to the transformed plant.

The “cold shock domain” (CSD) is a protein sequence that is homologousto the cold shock proteins. For the purposes of this invention, a coldshock domain containing protein is a “cold shock protein”. Greater than40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% amino acid identity is seenbetween E. coli CspA or B. subtilis CspB and the cold shock domains ofcold shock domain containing proteins (Wistow, Nature 344:823 (1990);Yamanaka, et al., Mol. Micro., 27:247, specifically see FIG. 1B in theYamanaka reference; Graumann, et al. TIBS 23:286).

As used herein “yeast” regularly refers to Saccharomyces cerevissiae butcould also include Schizosacchoramyces pombe and other varieties (fromthe genus Pichia, for example). “Corn” refers to Zea Mays and allspecies and varieties that can be bred with it. “Wheat” refers to all ofTriticum aestivum varieties including but not limited to spring, winter,and all facultative wheat varieties. “Wheat” includes any other wheatspecies, including but not limited to durum wheat (Triticum durum),spelt (Triticum spelta), emmer (Triticum dicoccum), and wild wheat(Triticum monococcum). “Wheat” also includes any species that can bebred with any of the aforementioned wheat species and offspring of saidcrosses (including triticale, a hybrid of wheat and rye). “Soybeans”refers to Glycine max or Glycine soja and any species or variety thatcan be bred with them. “Rice” refers to Oryza sativa and any species orvariety that can be bred with it. “Barley” refers to Hordeum vulgare andany species or variety that can be bred with it. “Oats” refers to Avenasativa and any species or variety that can be bred with it. “Canola” isa coined name recently given to seed, oil, and meal produced bygenetically modified rapeseed plants, oilseed rape (Brassica napus L.)and turnip rape (B. campestris L), herein canola includes all rapeseedplants and organisms that can be bred with them. E. coli and Escherichiacoli as used herein includes organisms of the Escherichia coli speciesand all strains of that this organism; i.e. E. coli K12. E. coli andEscherichia coli as used herein can also includes any organism that canconjugate with any E. coli strain when one is an F⁺ or Hfr strain, andthe other is not. B. subtilis and Bacillus subtilis refers to allorganism of the genus Bacillus, species subtilis. Agrobacteriumtumifaciens as used herein includes all strains and types of thisspecies. “Turf grasses” include all species and strains of grass everplanted, or that could be planted, to produce a turf, including but notlimited to; a lawn, a field for playing a game (i.e. football, baseball,or soccer), and all areas of a golf course (i.e. tee, fairway, green,rough, etc.). “Cotton” refers to all plants in the genus Gossypium andall plants that can be bred with them.

“Heat tolerance” is meant herein as a measure of a plants ability togrow under conditions where heat, or warmer temperature, woulddetrimentally affect the growth, vigor, yield, size, of the a plant ofthe same species. Heat tolerant plants grow better under conditions ofheat stress than non heat tolerant plants of the same species.

“Salt tolerance” refers to the ability of some plants to grow underosmotic stress, or stress caused by salts or ions in the water and soil.For example, a plant with increased growth rate, compared to a plant ofthe same species and/or variety, when watered with a liquid, or plantedin a media, containing a mix of water and ions that detrimentally affectthe growth of another plant of the same species would be said to be salttolerant. Some transformed plants have a greater tolerance for thesetypes of conditions than non-transformed plants of the same species andstrain.

All numbers used herein should be modified by the term “about”, aboutmeans that the number can vary, in either direction, by up to 10 percentand still retain the same meaning. For example, a 1 M solution shouldinclude all solutions of that type less than, and including, 1.1 M andmore than 0.9 M. For example, a percentage can also be modified, 10% isinclusive of all percentages from 9% to 11%. Terms defined by theadjective “exactly” are not defined by the term “about”.

A “glycine rich protein” is defined as a protein in a eukaryote that is,or has substantial identity with, or is a homologue of, a proteincontaining a cold shock domain.

“Survival after cold shock” is defined as the ability of a plant tocontinue growth for a significant period of time after being placed at atemperature below that normally encountered by a plant of that speciesat that growth stage. It should be noted that some plants, even those ofthe same species, have been selected for growth under cold conditions.The inbred Wigor strain of corn can tolerate cold conditions and has asignificantly higher survival rate when placed in those conditions thanmost commercial lines sold in the U.S. Wigor is sold commercially inPoland. Thus cold tolerance for transgenic plants must be comparedwithin plants of the same strain at the same relative age, as well asplants of the same species, to gain meaningful scientific data. Plantswould then be scored immediately, or some day(s) or week(s) later todetermine their viability, growth rate, and other phenotypes after theshock.

“Drought” or “water would be limiting for growth” is defined as a periodof dryness that, especially when prolonged, can cause damage to crops orprevent their successful growth. Again different plants of the samespecies, and those of different strains of the same species, may havedifferent tolerance for drought, dryness, and/or lack of water. In thelaboratory drought can be simulated by giving plants 95% or less waterthan a control plant and looking for differences in vigor, growth, size,root length, and myriad other physiologic and physical measures. Droughtcan also be simulated in the field by watering some plants, but notothers, and comparing their growth rate, especially where water isseverely limited for the growth of that plant.

Abiotic stress tolerance includes, but is not limited to, increasedyield, growth, biomass, health, or other measure that indicatestolerance to a stress which includes but is not limited to heat stress,salt stress, cold stress (including cold stress during germination),water stress (including but not limited to drought stress), nitrogenstress (including high and low nitrogen).

Biotic stress tolerance includes, but is not limited to, increasedyield, growth, biomass, health, or other measure that indicatestolerance to a stress which includes but is not limited to fungalinfection, bacterial infection, and viral infection of a plant.

Certain of the gene sequences disclosed as part of the invention arebacterial in origin, for example, certain prokaryotic cold shockproteins. It is known to one skilled in the art that unmodifiedbacterial genes are sometimes poorly expressed in transgenic plantcells. Plant codon usage more closely resembles that of humans and otherhigher organisms than unicellular organisms, such as bacteria. Severalreports have disclosed methods for improving expression of recombinantgenes in plants. These reports disclose various methods for engineeringcoding sequences to represent sequences which are more efficientlytranslated based on plant codon frequency tables, improvements in codonthird base position bias, using recombinant sequences which avoidsuspect polyadenylation or A/T rich domains or intron splicing consensussequences. While these methods for synthetic gene construction arenotable, the inventors have contemplated creating synthetic genes forcold shock proteins or proteins containing cold shock domains accordingto the method of Brown et al. (U.S. Pat. No. 5,689,052 1997, which isherein incorporated in its entirety by reference) and/or by the abovecited, as well as other methods. Thus, the present invention provides amethod for preparing synthetic plant genes express in planta a desiredprotein product. Briefly, according to Brown et al., the frequency ofrare and semi-rare monocotyledonous codons in a polynucleotide sequenceencoding a desired protein are reduced and replaced with more preferredmonocotyledonous codons. Enhanced accumulation of a desired polypeptideencoded by a modified polynucleotide sequence in a monocotyledonousplant is the result of increasing the frequency of preferred codons byanalyzing the coding sequence in successive six nucleotide fragments andaltering the sequence based on the frequency of appearance of thesix-mers as to the frequency of appearance of the rarest 284, 484, and664 six-mers in monocotyledonous plants. Furthermore, Brown et al.disclose the enhanced expression of a recombinant gene by applying themethod for reducing the frequency of rare codons with methods forreducing the occurrence of polyadenylation signals and intron splicesites in the nucleotide sequence, removing self-complementary sequencesin the nucleotide sequence and replacing such sequences withnonself-complementary nucleotides while maintaining a structural geneencoding the polypeptide, and reducing the frequency of occurrence of5′-CG-3′ dinucleotide pairs in the nucleotide sequence. These steps areperformed sequentially and have a cumulative effect resulting in anucleotide sequence containing a preferential utilization of themore-preferred monocotyledonous codons for monocotyledonous plants for amajority of the amino acids present in the desired polypeptide.Specifically all the protein mentioned herein are contemplated to bemade into synthetic genes as discussed above, or using similar methods,including but not limited to Escherichia coli CspA and Bacillus subtilisCspB.

The work described herein has identified methods of potentiating inplanta expression of cold shock proteins and proteins containing coldshock domains, which may confer resistance to many plant stresses, whichcan include but are not limited to cold, heat, drought, salt, and otherstresses, or stress related phenotypes (cold germination, survival aftercold stress, and other abiotic stresses) when ectopically expressedafter incorporation into the nuclear, plastid, or chloroplast genome ofsusceptible plants. U.S. Pat. No. 5,500,365 (specifically incorporatedherein by reference) describes a method for synthesizing plant genes tooptimize the expression level of the protein for which the synthesizedgene encodes. This method relates to the modification of the structuralgene sequences of the exogenous transgene, to make them more“plant-like” and therefore more likely to be translated and expressed bythe plant, monocot or dicot. However, the method as disclosed in U.S.Pat. No. 5,689,052 provides for enhanced expression of transgenes,preferably in monocotyledonous plants.

In developing the nucleic acid constructs of this invention, the variouscomponents of the construct or fragments thereof will normally beinserted into a convenient cloning vector, e.g., a plasmid that iscapable of replication in a bacterial host, e.g., E. coli. Numerousvectors exist that have been described in the literature, many of whichare commercially available. After each cloning, the cloning vector withthe desired insert may be isolated and subjected to furthermanipulation, such as restriction digestion, insertion of new fragmentsor nucleotides, ligation, deletion, mutation, resection, etc. so as totailor the components of the desired sequence. Once the construct hasbeen completed, it may then be transferred to an appropriate vector forfurther manipulation in accordance with the manner of transformation ofthe host cell.

A double-stranded DNA molecule of the present invention containing, forexample, a cold shock protein in an expression cassette can be insertedinto the genome of a plant by any suitable method. Suitable planttransformation vectors include those derived from a Ti plasmid ofAgrobacterium tumefaciens, as well as those disclosed, e.g., byHerrera-Estrella et al. (1983), Bevan (1984), Klee et al. (1985) and EPOpublication 120,516. In addition to plant transformation vectors derivedfrom the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternativemethods can be used to insert the DNA constructs of this invention intoplant cells. Such methods may involve, but are not limited to, forexample, the use of liposomes, electroporation, chemicals that increasefree DNA uptake, free DNA delivery via microprojectile bombardment, andtransformation using viruses or pollen.

A plasmid expression vector suitable for the introduction of a genecoding for a cold shock protein, or protein containing a cold shockdomain in monocots using electroporation could be composed of thefollowing: a promoter that functions in plants; an intron that providesa splice site to facilitate expression of the gene, such as the Hsp70intron (PCT Publication WO93/19189); and a 3′ polyadenylation sequencesuch as the nopaline synthase 3′ sequence (NOS 3′). This expressioncassette may be assembled on high copy replicons suitable for theproduction of large quantities of DNA.

An example of a useful Ti plasmid cassette vector for planttransformation is pMON-17227. This vector is described in PCTPublication WO 92/04449 and contains a gene encoding an enzymeconferring glyphosate resistance (denominated CP4), which is anexcellent selection marker gene for many plants. The gene is fused tothe Arabidopsis EPSPS chloroplast transit peptide (CTP2) and expressedfrom the FMV promoter as described therein. When an adequate numbers ofcells (or protoplasts) containing the sedoheptulose-1,7-bisphosphatasegene or cDNA are obtained, the cells (or protoplasts) are regeneratedinto whole plants. Choice of methodology for the regeneration step isnot critical, with suitable protocols being available for hosts fromLeguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot,celery, parsnip), Cruciferae (cabbage, radish, canola/rapeseed, etc.),Cucurbitaceae (melons and cucumber), Gramineae (wheat, barley, rice,maize, etc.), Solanaceae (potato, tobacco, tomato, peppers), variousfloral crops, such as sunflower, and nut-bearing trees, such as almonds,cashews, walnuts, and pecans.

Plants that can be made to express cold shock proteins by practice ofthe present invention include, but are not limited to, Acacia, alfalfa,aneth, apple, apricot, artichoke, arugula, asparagus, avocado, banana,barley, beans, beet, blackberry, blueberry, broccoli, brussels sprouts,cabbage, canola, cantaloupe, carrot, cassava, cauliflower, celery,cherry, cilantro, citrus, clementines, coffee, corn, cotton, cucumber,Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs,gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks,lemon, lime, Loblolly pine, mango, melon, mushroom, nut, oat, okra,onion, orange, an ornamental plant, papaya, parsley, pea, peach, peanut,pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate,poplar, potato, pumpkin, quince, radiata pine, radicchio, radish,raspberry, rice, rye, sorghum, Southern pine, soybean, spinach, squash,strawberry, sugarbeet, sugarcane, sunflower, sweet potato, sweetgum,tangerine, tea, tobacco, tomato, turf, a vine, watermelon, wheat, yams,zucchini, or any other plant.

“Promoter” refers to a DNA sequence that binds an RNA polymerase (andoften other transcription factors) and promotes transcription of adownstream DNA sequence. Promoters are often provide enhanced or reducedexpression in some tissues when compared to others. Promoter selection,specifically selecting promoters that increase expression when a plantis undergoing abiotic stress could be particularly useful in the instantinvention.

It has been observed in the art that some stress responses have similareffects on the plant, and resistance to one may provide resistance toanother. This is seen, for example, between the responses to dehydrationand low temperature (Shinozaki, et al., Current Opinions in PlantBiology 3(3):217, 2000). Many other papers show the generalinterrelationship between different abiotic stresses, and might indicatethat tolerance to one stress might lead to greater tolerance of severalother abiotic stresses (Pernas, et al., FEBS Lett 467(2-3):206, 2000;Knight, Int Rev Cytol 195:269, 2000; Didierjean, et al., Planta 199: 1,1996; Jeong, et al., Mol Cells 12:185, 2001).

Expression cassettes and regulatory elements found in the DNA segmentoutside of the plant expression elements contained in the T-DNA arecommon in many plasmid DNA backbones and function as plasmid maintenanceelements, these include, but are not limited to, the aad (Spc/Str) genefor bacterial spectinomycin/streptomycin resistance, the pBR322 ori(ori322) that provides the origin of replication for maintenance in E.coli, the born site for the conjugational transfer into theAgrobacterium tumefaciens cells, and a DNA segment is the 0.75 kb oriVcontaining the origin of replication from the RK2 plasmid. In addition,those plasmids intended for transformation into plants often contain theelements necessary for the endogenous DNA integration proteins ofAgrobacterium to function to insert the element. These include borders(right (RB) and left (LB) borders).

The laboratory procedures in recombinant DNA technology used herein arethose well known and commonly employed in the art. Standard techniquesare used for cloning, DNA and RNA isolation, amplification andpurification. Generally enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like are performedaccording to the manufacturer's specifications. These techniques andvarious other techniques are generally performed according to Sambrooket al., Molecular Cloning—A Laboratory Manual, 2nd. ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1989).

All publications and published patent documents cited in thisspecification are incorporated herein by reference to the same extent asif each individual publication or patent application is specifically andindividually indicated to be incorporated by reference.

The following examples are included to demonstrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings and examples is to be interpreted as illustrative and not in alimiting sense.

EXAMPLES Example 1

pMON57396 (FIG. 1) is a binary vector for Agrobacterium-mediatedtransformation and constitutive expression of a protein (SEQ ID NO: 56)similar to Escherichia coli CspA in Arabidopsis. To clone the E. coliCspA gene, two gene specific primers, MF1 and MF2, were designed basedon the CspA sequence information (Genbank M30139, GI:409136) from theNational Center for Biotechnology Information, which is part of theNational Library of Medicine, in turn part of the National Institutes ofHealth (NCBI). The sequence for MF1 is AGGTAATACACCATGGCCGGTAA (SEQ IDNO: 66), which anneals at the translational start site of CspA andintroduces an NcoI site at the 5′ end, while the sequence of MF2 isTTAAGCAGAGAATTCAGGCTGGTT (SEQ ID NO: 67), which anneals at the lastcodon of CspA and introduces an EcoRI site at the end of the primer. PCRwas performed to isolate E. coli CspA. Specifically, E. coli DH5a cellswere lysed and a small amount of the lysate was used as a template toamplify the CspA gene using MF1 and MF2 primers, Taq polymerase anddNTPs from Roche Molecular Biochemicals (Indianapolis, Ind.). Thethermal cycling conditions were as follows: 94° C., 1 min, followed by30 cycles of 94° C., 16 seconds; 55° C., 1 min and 72° C., 1 min. Theamplified CspA DNA was purified by gel-electrophoresis, digested withNcoI and EcoRI and ligated to a binary vector pMON23450 (FIG. 2) thathad previously been linearized by digestion with NcoI and EcoRI.Ligation was performed using T4 ligase and following proceduresrecommended by the manufacturer (BRL/Life Technologies, Inc.,Gaithersburg, Md.). The ligation mix was transformed into E. coli cellsfor plasmid propagation (Sambrook et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor Press, 1989). Thetransformed cells were plated on appropriate selective media (Sambrooket al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, ColdSpring Harbor Press, 1989) and colonies were scored hours or days later.Plasmids were prepared from individual colonies and full-insert sequencewas determined.

The resulting plasmid was also confirmed by restriction mapping (forexample, see Griffiths, et al, An Introduction to Genetic Analysis,6^(th) Edition pp 449-451, ISBN 0-7167-2604-1, W.H. Freeman and Co., NewYork) and sequencing. As the chosen NcoI-EcoRI cloning site in thevector was flanked by a CaMV e35S promoter at the upstream (5′) and anepitope tag (Flag, which encodes the oligopeptide DYKDDDK (SEQ ID NO:68), SIGMA, St Louis) at the downstream (3′), the E. coli CspA in thisconstruct is thus tagged at the C-terminus by the Flag epitope tag andwill be driven transcriptionally by the CaMV e35S promoter upontransformation in Arabidopsis. The above cloning results in a plasmidencoding a protein similar to SEQ ID NO: 55. The resulting plasmid iscalled pMON57396.

Example 2

pMON57397 (FIG. 2) is a binary vector for Agrobacterium-mediatedtransformation and constitutive expression of a protein (SEQ ID NO: 57),like Escherichia coli CspA protein, in Arabidopsis. To create pMON57397,the binary vector pMON57396 containing the Escherichia coli CspA gene(see example above) tagged at the C-terminus by the Flag epitope tag,was digested with restriction enzymes XhoI and SalI to cleave thesesites in the vector and release the FLAG epitope tag (The FLAG tagencodes the oligopeptide DYKDDDK, SIGMA, St Louis). The linearizedplasmid was then purified and religated. Ligation was performed using T4ligase and following procedures recommended by the manufacturer(BRL/Life Technologies, Inc., Gaithersburg, Md.). The ligation mix wastransformed into E. coli cells for plasmid propagation (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Press, 1989). The transformed cells were plated on appropriateselective media (Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, Cold Spring Harbor Press, 1989) and colonieswere scored hours or days later. Plasmids were prepared from individualcolonies and full-insert sequence was determined. The cloning aboveresults in the creation of a plasmid encoding a protein similar to SEQID NO: 57.

The resulting plasmid was also confirmed by restriction mapping toensure that XhoI and SalI sites were absent (for example, see Griffiths,et al, An Introduction to Genetic Analysis, 6^(th) Edition pp 449-451,ISBN 0-7167-2604-1, W.H. Freeman and Co., New York) and sequencing. TheE. coli CspA gene in this construct is untagged at the C-terminus and isdriven transcriptionally by the CaMV e35S promoter.

Example 3

pMON57398 (FIG. 4) is a binary vector for Agrobacterium-mediatedtransformation and constitutive expression of a protein (SEQ ID NO: 59)like Bacillus subtilis CspB, in Arabidopsis. To clone the B. subtilisCspB gene, two gene-specific primers, MF3 and MF4a, were designed basedon the CspB sequence information (Genbank U58859, gi:1336655) from theNational Center for Biotechnology Information, which is part of theNational Library of Medicine, in turn part of the National Institutes ofHealth (NCBI). The sequence for MF3 is AGGAGGAAATTCCATGGTAGAAG (SEQ IDNO: 69), which anneals at the translational start site of CspB andintroduces an NcoI site at the 5′ end, while the sequence of MF4a isTCAATTTATGAATTCGCTTCTTTAGT (SEQ ID NO: 70), which anneals at the lastcodon of CspB and introduces an EcoRI site at the end of the primer. PCRwas performed to isolate B. subtilis CspB. Bacillus subtilis cells wereobtained from Carolina Biological Supply (Burlington, N.C.), the cellswere lysed and a small amount of the lysate was used as a template toamplify the CspB gene using MF3 and MF4a primers, Taq polymerase anddNTPs from Roche Molecular Biochemicals. The thermal cycling conditionswere as follows: 94° C., 1 min, followed by 30 cycles of 94° C., 16seconds; 55° C., 1 min and 72° C., 1 min. The amplified CspB DNA waspurified by gel-electrophoresis, digested with NcoI and EcoRI andligated to a binary vector pMON23450 (FIG. 5) that had previously beenlinearized by digestion with NcoI and EcoRI. Ligation was performedusing T4 ligase and following procedures recommended by the manufacturer(BRL/Life Technologies, Inc., Gaithersburg, Md.). The ligation mix wastransformed into E. coli cells for plasmid propagation. The transformedcells were plated on appropriate selective media (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor Press, 1989) and colonies were scored a day later. Plasmids wereprepared from individual colonies and full-insert sequence wasdetermined.

The resulting plasmid was also confirmed by restriction mapping (forexample, see Griffiths, et al, An Introduction to Genetic Analysis,6^(th) Edition pp 449-451, ISBN 0-7167-2604-1, W.H. Freeman and Co., NewYork) and sequencing. As the chosen NcoI-EcoRI cloning site in thevector was flanked by a CaMV e35S promoter at the upstream (5′) and anepitope tag (Flag, which encodes the oligopeptide DYKDDDK (SIGMA, StLouis) at the downstream (3′), the B. subtilis CspB like gene in thisconstruct is thus tagged at the C-terminus by the Flag epitope tag andwill be driven transcriptionally by the CaMV e35S promoter upontransformation in Arabidopsis. This cloning results in a plasmid withthe sequence encoding a protein similar to SEQ ID NO: 59 being insertedinto said plasmid.

Example 4

pMON57399 (FIG. 6) is a binary vector for Agrobacterium-mediatedtransformation and constitutive expression of a protein (SEQ ID NO: 61)like Bacillus subtilis CspB in Arabidopsis. To create pMON57399, thebinary vector pMON57398 containing the Bacillus subtilis CspB gene (seeexample above) tagged at the C-terminus by the Flag epitope tag, wasdigested with restriction enzymes XhoI and SalI to cleave these sites inthe vector and release the FLAG epitope tag (The FLAG tag encodes theoligopeptide DYKDDDK, SIGMA, St Louis). The linearized plasmid was thenpurified and religated. Ligation was performed using T4 ligase andfollowing procedures recommended by the manufacturer (BRL/LifeTechnologies, Inc., Gaithersburg, Md.). The ligation mix was transformedinto E. coli cells for plasmid propagation (Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) Edition, Cold Spring Harbor Press,1989). The transformed cells were plated on appropriate selective media(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor Press, 1989) and colonies were scored hoursor days later. Plasmids were prepared from individual colonies andfull-insert sequence was determined. This cloning results in a plasmidwith a sequence encoding a protein similar to SEQ ID NO: 61 beinginserted into said plasmid.

The resulting plasmid was also confirmed by restriction mapping toensure that XhoI and SalI sites were absent (for example, see Griffiths,et al, An Introduction to Genetic Analysis, 6th Edition pp 449-451, ISBN0-7167-2604-1, W.H. Freeman and Co., New York) and sequencing. As thechosen NcoI-EcoRI cloning site in the vector was flanked by a CaMV e35Spromoter at the upstream (5′) N-terminus, the B. subtilis CspB gene inthis construct is untagged at the C-terminus and is driventranscriptionally by the CaMV e35S promoter upon transformation inArabidopsis. Said plasmids were transformed into Agrobacteriumtumefaciens.

Example 5

Arabidopsis plants may be transformed by any one of many availablemethods. For example, Arabidopsis plants may be transformed using Inplanta transformation method by vacuum infiltration (see, Bechtold etal., In planta Agrobacterium mediated gene transfer by infiltration ofadult Arabidopsis thaliana plants. CR Acad. Sci. Paris Sciences de lavie/life sciences 316: 1194-1199 (1993). This example illustrates howArabidopsis plants may be transformed.

Stock Plant Material and Growth Conditions

Prepare 2.5 inch pots with soil and cover them with a mesh screen,making sure that the soil is not packed too tightly and the mesh is incontact with the soil surface (this ensures that the germinatingseedlings will be able to grow through the mesh). Sow seeds and coverwith a germination dome. Vernalize seeds for 3-4 days. Grow plants underconditions of 16 hours light/8 hours dark at 20-22° C., 70% humidity.Water twice weekly, and fertilize from below with ½×(half of thestrength recommended by the manufacturer) Peters 20-20-20 fertilizer(from Hummert International, Earth City, Mo.). Add micronutrients(Hummert's Dyna-grain Soluble Trace Elements) (in full strengthrecommended by the manufacturer) every other week. After about 1-2weeks, remove the dome and thin the pots to one or two plants per pot.Clip the primary bolt, when it develops, to encourage more secondarybolt formation. In 5-7 days the plants will be ready for infiltration.

Agrobacterium Preparation (Small scale and Large scale cultures):

Agrobacterium strain ABI is streaked onto an LB plate containingSpectinomycin 100 mg/L, Streptomycin 100 mg/L, Chloramphenicol 25 mg/L,and Kanamycin 50 mg/L (denoted SSCK). Two days prior to infiltration, aloop of Agrobacterium is placed into a tube containing 10 mls LB/SSCKand put on a shaker in the dark at 28° C. to grow overnight. Thefollowing day, the Agrobacterium is diluted 1:50 in 400 mis YEP/SSCK andput on a shaker at 28° C. to grow for 16-20 hours. (Note: we have foundthe transformation rate is significantly better when LB is used for thefirst overnight growth and YEP is used for the large scale overnightculture).

Infiltration

Harvest the Agrobacterium cells by pouring into a 500 ml centrifugebottle and spinning at 3500 rpm for 20-25 minutes. Pour off thesupernatant. Dry the pellet and then resuspend in 25 ml InfiltrationMedium (MS Basal Salts 0.5%, Gamborg's B-5 Vitamins 1%, Sucrose 5%, MES0.5 g/L, pH 5.7) with 0.44 nM benzylaminopurine (BAP) (10 μl of a 1.0mg/L stock in DMSO per liter) and 0.02% Vac-In-Stuff (Silwet L-77) fromLehle Seeds (Round Rock, Tex.). The BAP and Silwet L-77 are added freshthe day of infiltration. Add 200 μl of Silwet L-77, and 20 μl of BAP(0.5 mg/L stock). Using Infiltration Medium as your blank, take theOD₆₀₀ of a 1:10 dilution of the Agrobacterium suspensions. Calculate thevolume needed for 400 ml of Agrobacterium suspension/infiltrationmedium, OD600=0.6, for the vacuum infiltration.

${{Equation}\text{:}\mspace{14mu}\frac{\left( {{final}\mspace{14mu}{volume}} \right)*\left( {{final}\mspace{14mu}{OD}\; 600} \right)}{{OD}\; 600}} = {{Volume}\mspace{14mu}{needed}\mspace{14mu}{for}\mspace{14mu}{final}\mspace{14mu}{OD}\; 600\mspace{14mu}{of}\mspace{14mu} 0.6}$

Place resuspended culture in a Rubbermaid container inside a vacuumdessicator. Invert pots containing plants to be infiltrated into thesolution so that the entire plant is covered, including the rosette, butnot too much of the soil is submerged. Soak the plants with water for atleast 30 min. prior to infiltration. (This keeps the soil from soakingup the Agrobacterium suspension).

Draw a vacuum of ˜23-27 in. Hg for 10 min. Quickly release the vacuum.Briefly drain the pots, place them on their sides in a diaper-linedtray, cover the tray with a dome to maintain humidity, and return togrowth chamber. The following day, uncover the pots, set them upright,and remove the diaper. Do not water plants for ˜5 days. After the 5 daysare up, allow the plants to be watered and to continue to grow under thesame conditions as before. (The leaves that were infiltrated maydegenerate but the plant should survive until it is finished flowering).

Harvesting and Sterilizing Seed

Cone the plants, individually, by using the Lehle Aracons (Lehle Seeds,Round Rock, Tex.) approximately 2 weeks after infiltration. After all ofthe seed is matured and has set (˜4 weeks post-infiltration), remove theplants from water to dry down the seeds. Approximately 2 weeks laterharvest the seeds by cutting the branches below the cone. Clean the seedby using a sieve to catch the silique and branch material and allow theseed to go through. Place the seed in an envelope or in 15 ml conicaltubes.

Transfer desired amount of seeds to 15 ml conical tubes prior tosterilization. Loosen the lid to the conicals and place them on theirside in a vacuum dessicator with a beaker containing 400 ml of bleachClorox (Clorox Company, Oakland, Calif.) and 4 ml of Hydrochloric Acid.(Add the HCl to the Clorox in a fume hood). Pull a vacuum just to sealthe dessicator, and close the suction (i.e. so that the dessicator isstill under a vacuum but the vacuum is not still being directly pulled)for ˜16 hrs. After sterilization, release the vacuum and place tubescontaining seed in a sterile hood (keep caps loose so gas can still bereleased).

Plate (“sprinkle”) the seed on selection plates containing MS BasalSalts 4.3 g/L, Gamborg'a B-5 (500×) 2.0 g/L, Sucrose 10 g/L, MES 0.5g/L, and 8 g/L Phytagar (Life Technologies, Inc., Rockville, Md.) withCarbenicillin 250 mg/L, Cefotaxime 100 mg/L. Selection levels willeither be kanamycin 60 mg/L, Glyphosate 60 μM, or Bialaphos 10 mg/L.

A very small amount of seed can be first plated out to check forcontamination. If there is contamination, re-sterilized seeds for ˜4more hours and check for contamination again. The second sterilizationis usually not necessary, but sometimes the seed harbors a fungalcontaminant and repeat sterilizations are needed. (The sterilizationduration generally is shorter than 16 hours because of significantlydecreased germination rates starting at 24 hr. sterilization duration).Seal plates with parafilm and place in a cold room to vernalize for ˜2-4days. After seeds are vernalized, place in percival with cool whitebulbs.

Transfer to Soil

After 5-10 days at ˜26° C. and a 16/8 light cycle, the transformantswill be visible as green plants. After another 1-2 weeks, plants willhave at least one set of true leaves. Transfer plants to soil, coverwith a germination dome, and move to a growth chamber with normalArabidopsis growth conditions. Keep covered until new growth is apparent(usually 5-7 days).

Example 6

In order to compare the growth of wildtype non-transgenic and CspA orCspB transgenic Arabidopsis plants, verticle growth was allowed insterile Petri dishes:

Wildtype or transgenic seeds were liquid sterilized using the followingmethod:

-   -   5 minute incubation in 70% ethanol following vortex mixing    -   5 minute incubation in 30% Chlorox (6.15% sodium        hypochlorite)+0.01% Triton X-100 following vortex mixing    -   5 consecutive sterile water washes

Seeds were plated onto plastic, 100×15 mm square petri dishes (BectonDickinson-Falcon #35-1112), each containing 40 ml of agar media made asfollows:

0.5× Murashige and Skoog media with macronutrients, micronutrients andvitamins (Sigma #M5519), adjusted to pH 5.8 with ammonium hydroxide andcontaining 1% Phytagel (Sigma # P8169) for solid support.

Ten wild type Arabidopsis seeds were plated across one half of a petridish, approximately 1 cm from the edge and evenly spaced. This was donewith a Gilson P-200 Pipetteman using sterile tips. Ten CspA or CspBtransgenic Arabidopsis seeds were similarly plated across the other halfof the petri dish, evenly spaced. The plates were labeled with a markingpen to indicate which half contained the transgenic seeds.

The petri dishes were put at 4° C. for 3 days in the dark to stratifythe seeds and then placed in a Percival incubator (model AR-36L) at 8°C. for 6 weeks at 24 hour constant light of 120 microeinsteins/squaremeter. At the end of this incubation, the size of the CspA and CspBrosettes were compared to that of wildtype and found to be larger. Thiscan be seen in FIG. 16. This can be seen in the first, second, and lastpictured plate where the above assay was used. In FIG. 16, the thirdpicture (CspB+Flag, pMON57399) displays a plate wherein the plants wereput through a cold shock assay similar to that described below.

Cold Shock Seedling Vigor Assessment of Transgenic Arabidopsis thalianaSeeds: Horizontal Plate Assay.

Introduction

This is a procedure for assessing the ability of transgenic Arabidopsisseeds that have germinated at normal temperatures on media agar inhorizontal petri plates to continue to grow upon a shift to chilling. Inshort, seeds from control plants and seeds from tester transgenic plantsare sterilized, stratified, and plated in 6×8 grids on either half of apetri dish. The plate is incubated at normal temperature in a horizontalposition for one week and then shifted to chilling temperature for twoadditional weeks, maintaining the horizontal position of the plate. Thecanopy area of seedlings is recorded by digital photography andquantitated using imaging software. The ratio of the total canopy areaof the tester seedlings to that of the control seedlings can be used asa quantitative parameter to compare the cold tolerance potential ofvarious genes of interest in transgenic tester lines.

Materials: the following assumes the normal capital equipment availablein a standard biotechnology laboratory (autoclave, balance, laminar flowhood, etc.)

-   -   Arabidopsis seed: the protocols here have been used with        Arabidopsis thaliana cv. Columbia, but ought to be suitable for        other Arabidopsis species as well.    -   Petri dishes: Falcon #35-1112 (100 mm square×15 mm deep)    -   Media: Sigma M5519=Murashige & Skoog Basal Media    -   Phytagel (Sigma #P-8169)    -   1-liter glass bottles in which to autoclave media agar and from        which to pour plates. We use Corning glass bottles with the        orange screw caps.    -   Magnetic stirrers and magnetic stir bars    -   Electric pipettor usable with 50 ml plastic pipettes.    -   Small fluorescent light box with plastic magnifying tense for        plating seeds.    -   P1000 Gilson pipetor (or equivalent) and sterile tips    -   P200 Gilson pipetor (or equivalent) and sterile tips    -   70% Ethanol, sterile    -   30% Chlorox bleach+0.1% Tween 20    -   Sterile filtered deionized water    -   Sterile microcentrifuge tubes and tube racks    -   4° C. cold room, cold box or refrigerator, preferably dark    -   22 degrees C. Percival plant growth chamber or equivalent with        ˜150 μE/m²/sec light source    -   8 degrees C Percival plant growth chamber or equivalent with        ˜150 μE/m²/sec light source    -   Semipermeable surgical tape 3M Micropore tape (3M #1530-1)    -   Black (Sharpie) marker    -   Vacuum aspirator with trap    -   Glassine balance weighing paper (VWR #12578-165)    -   Calculator    -   Notebook    -   IBM compatible computer    -   Image-Pro Plus software, version 4.1.0.0    -   Microsoft Excel software        Protocol:    -   1—Aliquot seeds for storage vials or envelopes to sterile        microcentrifuge tubes    -   2—Label tubes with sharpie to retain identity of seeds    -   3—Surface sterilize seeds in tubes by successive washing with        the following solutions and waiting times listed below. Note,        invert tubes during washings at least twice to ensure good        surface contact of solutions on seeds. Seeds will fall down to        the bottom of the tube, making a soft pellet:        -   a. 70% Ethanol, sterile, for 3 to 5 minutes        -   b. 30% Chlorox bleach+0.1% Tween 20, for 3 to 5 minutes        -   c. Sterile filtered deionized water, for 30 seconds        -   d. Repeat c. four more times and on the last time, leave            ˜0.5 ml of sterile water remaining over the seed pellet.    -   1—Place microcentrifuge tubes in the dark at 4° C. for three        days to stratify the seeds for more uniform germination upon        plating.

[Alternatively, the seeds can be directly plated onto media agar petridishes, taped sealed and the petri dish can be put at 4° C. in the darkfor three days prior to the 8° C. cold incubation—see below]

-   -   2—Make plates by preparing 1-liter aliquots of 0.5× Murashige        and Skoog media in the glass bottles, adjust pH to 5.8 with        ammonium hydroxide, then add 10 grams of Phytagel. Use a        magnetic stirrer when adjusting the pH and to mix in the        phytagel uniformly, then autoclave on liquid setting (slow        exhaust) for 45 minutes.    -   3—Pour plates in the laminar flow hood using the electric        pipettor with the 50 ml sterile pipette to deliver 40 ml of        media to each plate, immediately covering the plate with the        lid.    -   4—Allow plates to cool in laminar flow hood for at least 2 hours        with the blower off and store in dated plastic bags at 4° C.    -   5—Label plates and plate seeds:    -   1—Tape all four edges of the plate with semipermeable micropore        tape, label with the date and put plates in a Percival incubator        set at 22 C and 16 hour day light cycle at ˜100 μE/m² sec. Place        the plates in a horizontal position only one layer thick and        incubate for 7 days. Photograph each plate with a digital camera        and store the data to a compact disk.    -   2—Transfer plates to a Percival incubator set at 8° C. and 24        hour day light cycle at ˜100 μE/m² sec, Place the plates in a        horizontal position only one layer thick and incubate for up to        3 additional weeks. Photograph each plate with a digital camera        and store the data to a compact disk.    -   3—Observe plates every 2 to 3 days to see how tester germplasms        are proceeding compared to controls and digitally photograph at        times that are representative of the general performance of the        germplasms. This should take less than 2 weeks (3 weeks at the        most) of incubation at 8° C. Those germplasms that take longer        to show a difference need to be plated at a lower seed density        to avoid overcrowding at the time the digital photograph is        taken.    -   4—Measure rosette canopy area using digital camera photography        and Image-Pro Plus software. Calculate the average seedling        canopy for control and tester populations, eliminating seeds        from the analysis that never germinated. Calculate the ratio        between the average seedling canopy area post temperature shift        for the control seedlings and the tester seedlings, the standard        deviation and standard error for control and tester seedling        sets. Ascertain if there is a statistical difference between the        tester seedlings and the control seedlings. Record results in a        notebook.    -   5—Discard plates and seedlings in appropriate disposal        containers for transgenic plant materials (gray bins with clear        plastic waste bags).

Example 7

PCR products of the CspA and CspB genes were ligated to vector pCR-TOPO2.1 according to the manufacturer's protocol (Invitrogen, Carlsbad,Calif.). The NcoI/EcoRI fragments of the pCR-TOPO 2.1 derivatives weresubcloned into pMON48421 (FIG. 7), linearized by the same restrictionenzymes. The NotI fragments of the pMON48421 derivatives encompassingthe 35S promoter, Csp genes, and the e9 terminator were subcloned intopMON42916 (FIG. 17) at the NotI site to create pMON56609 (FIG. 8) andpMON56610 (FIG. 9) which contain the CspA and CspB genes, respectively.Said plasmids were transformed into Agrobacterium tumefaciens by knownmethods. pMON56609 is thought to contain a nucleotide sequence encodinga protein similar to SEQ ID NO: 7. pMON56610 is thought to contain anucleotide sequence encoding a protein similar to SEQ ID NO: 9.

Example 8 Agrobacterium Preparation

Agrobacterium strain EHA105 is streaked on LB plate containing Kanamycin50 mg/L and Hygromycin 50 mg/L (denoted LB/KH). Two days prior toco-cultivation, a loop of Agrobacterium is transferred to a tubecontaining 10 ml LB/KH and incubated on a shaker in dark at 28 C for 24hours. This culture is diluted to 1:100 in 20 ml LB/KH and incubated ona shaker in dark at 28 C overnight. The following day 1 ml of 1:2dilution of this culture is taken in a cuvette and OD600 is taken withLB/KH as blank. Calculate the volume needed for 5 ml of agrobacteriumsuspension of O.D 1.0 for co-cultivation.

${{Equation}\text{:}\mspace{14mu}\frac{\left( {{final}\mspace{14mu}{volume}} \right)*\left( {{final}\mspace{14mu}{OD}\; 600} \right)}{{OD}\; 600}} = {{Volume}\mspace{14mu}{needed}\mspace{14mu}{for}\mspace{14mu}{final}\mspace{14mu}{OD}\; 600\mspace{14mu}{of}\mspace{14mu} 1.0}$

Take the required volume of agrobacterium culture in a 40 ml centrifugetube and spin at 7000 rpm for 7 minutes. Discard the supernatant and drythe pellet. Resuspend the pellet in 5 ml of co-cultivation media (CCMEDIA-MS Basal salts, Sucrose 20 g/L, Glucose 10 g/L, thiamine HCl 0.5mg/L, L-Proline 115 mg/L, 2,4-D 2 mg/L) with 20 mg/L of acetosyringone.

Transformation of Rice Embryos:

Panicles were harvested from greenhouse grown Nipponbare and Taipai 309rice varieties. The panicles were sterilized by immersing in 50%commercial bleach for 10 minutes followed by rinsing in steriledistilled water. The panicles were given a 70% alcohol treatment for 3mins. The seeds were then removed from the panicles and dehuskedindividually and transferred to a falcon tube containing 0.1% tween 20solution. The seeds were then treated with 70% alcohol in the laminarair flow chamber. Then the seeds were rinsed with sterile water. Thiswas followed by a 50% bleach treatment for 45 minutes. The seeds wererinsed 5 times in sterile distilled water. Finally the seeds are given0.1% mercuric chloride treatment for 5 minutes. The seeds were againwashed 8 times with sterile distilled water.

The embryos were excised aseptically from the sterile seeds in thelaminar flow chamber and placed on solid co-cultivation media (CC MEDIAwith 2 g/L phytagel). 50 μL drops of the agrobacterium suspension wereplaced on a sterile petri-plate. 10 embryos were transferred to eachdrop. The infection was allowed for 15 minutes. The agrobacteriumsuspension was removed with a sterile pipette tip. The infected embryoswere transferred to a fresh solid CC MEDIA plate and kept in dark for 2days. On the third day the embryos were washed with cefotaxime 500 mg/L.The embryos were then dried on sterile filter paper and placed on Delaymedia (MS Basal salts, Thiamine HCl 1 mg/L, Glutamine 500 mg/L,Magnesium Chloride 750 mg/L, casein hyrolysate 100 mg/L, Sucrose 20mg/L, 2,4-D 2 mg/L, Pichloram 2.2 mg/L, Cefotaxime 250 mg/L). theembryos are kept on delay medium in dark for a period of 7 days. Duringthis period calli are formed. The calli are transferred to selectionmedia (Delay medium with 50 mg/L Hygromycin) and stored in dark for 10days. The calli are sub-cultured to fresh selection media after this 10day period. After another 10 days the calli are transferred toregeneration media (MS Basal salts, sucrose 30 mg/L, Kinetin 2 mg/L, NAA0.2 mg/L, Cefotaxime 250 mg/L, hygromycin 25 mg/L) and kept in dark for7 days. The calli are then transferred to fresh regeneration media andmoved to a 16-hour photoperiod at 30 C. The shoots developed on thiscallus are transferred to rooting media (half strength MS Basal salts,sucrose to 15 g/L, Cefotaxime 250 mg/L, Hygromycin 25 mg/L). The rootedshoots are transferred to test-tubes containing water and placed in amist chamber for hardening.

Plants were selected as positive. This could be done, for example, usingmethods similar to those described in examples 12-14, and 26-29.Including breeding methods described to create the next generation oftransgenic plants.

Example 9 Cold Stress Response at Three Leaf Stage CspB and CspA RiceTransgenic Plants

Plant Material Preparation:

Germination: Seeds were sterilized by treating with 0.01 percentmercuric chloride for 3 minutes and washed thoroughly for ten times inmilique water to remove the traces of mercuric chloride. Sterilizedseeds were allowed to imbibe by soaking in milique water for 3 hours.The imbibed seeds were germinated on a sterilized moist filter paper at30° C. temperature and 60% RH using a seed germinator (SerwellInstruments Inc.).

Establishment of three leaf stage seedlings: The three day oldgerminated seedlings were transferred to portrays (52.5 mm (length)×26mm (depth)×5.2 mm (diameter)) in the greenhouse having light intensityof 800 micro mol./mt2/sec. and 60% RH. The seedlings were grown tillthree-leaf stage (Approximately for 12 days) in portrays containing redsandy loam soil. Fertilizer solution was applied to the seedlings once aweek till the completion of the experiments (N-75 PPM, P-32 PPM, K-32PPM, Zn-8 PPM, Mo-2 PPM, Cu-0.04 PPM, B-0.4 PPM and Fe-3.00 PPM).

CspB-R2 Plant Analysis

Protocol: Three leaf stage rice seedlings (12 day old) were subjected toa cold stress of 10° C. for 4 days in presence of 100 micromol./mt²/sec. light and 70% RH (Percival growth chamber). After thestress treatment the plants were allowed to recover in the greenhousefor 10 days and on the 10^(th) day the growth observations for survivedplants and photographic evidences were recorded. Each treatment had 10replications per line and they were completely randomized.

Results: Among eight different lines tested for cold stress tolerancesix lines exhibited significantly higher cold tolerance compared to thewild type. The lines including R2-226-6-9-3, R2-226-29-1-1,R2-257-20-2-1, R2-238-1-1-3, R2-230-4-4-2 and R2-257-3-1-3 showed highcold tolerance by exhibiting high recovery growth and less percentreduction in growth (over non-stressed control) compared to the wildtype (table-1, plate-1). The line R2-230-4-42, has performed extremelywell, it exhibited 100 percent survival and maintained good growthduring recovery (Table 1).

TABLE 1 Three leaf stage cold stress recovery growth observations ofCspB R2 transgenic lines. % % Reduction in Survival at plant height endof Plant height (cm) over non- Lines recovery Stressed Non-stressedstressed R2-257-17-1-1 13 21.5 ± 11  43.44 ± 4.09 50.38 R2-230-34-1-2 5320.78 ± 6.3   45.0 ± 3.51 53.82 R2-226-6-9-3 60 27.5 ± 7   33.74 ± 4.6518.49 R2-226-29-1-1 53  27.6 ± 10.7 35.22 ± 4.06 21.63 R2-257-20-2-1 9332.39 ± 5.48  44.0 ± 2.95 27.27 R2-238-1-1-3 80 29.25 ± 8.19 40.72 ±5.8  25 R2-230-4-4-2 100 33.95 ± 4.10   45 ± 3.98 24 R2-257-3-1-3 4029.80 ± 2.66   42 ± 4.11 28.5 WT - Taipei 26 23.93 ± 5.61 45.0 ± 3.746.6 (Index: WT = Wild type)CspB-R3 Plant Analysis

Protocol: Three leaf stage seedlings were exposed to cold stress of 8degree Celsius for 1 day in presence of 1000 micro mol./mt2/sec. oflight. Later the seedlings were allowed to recover at 28 degree Celsiusin the greenhouse for 15 days and at the end of recovery the plantheight was recorded.

Results: Eight different lines tested for cold stress tolerance and allthe eight lines showed improved tolerance compared to wild type(non-transgenic) plants. These results confirmed the R2 analysis datashowing improved cold tolerance (Table 2).

TABLE 2 Three leaf stage cold stress recovery growth observations ofCspB R3 transgenic lines. Percent reduction Non-stressed in plantStressed-plant plant height height height (cm) at end of (cm) at endover Lines recovery of recovery non-stress R3-226-6-9-3  28.8 ± 2.8829.34 ± 7.20 1.84 R3-226-29-1-3-4 30.18 ± 3.19 32.07 ± 3.79 5.89R3-230-4-4-2-1 30.42 ± 2.16 35.09 ± 4.19 13.30 R3-230-34-1-2-1 32.14 ±3.41  37.4 ± 5.68 14.01 R3-238-1-1-3-4 29.54 ± 3.61  32.2 ± 3.56 8.26R3-257-3-1-3-1 27.12 ± 3.38 30.86 ± 3.82 12.11 R3-257-15-1-1-2 23.84 ±2.85 26.71 ± 1.92 10.74 R3-257-20-2-1-1  33.8 ± 3.48 38.82 ± 1.97 12.93WT - Taipei  23.9 ± 3.74 36.65 ± 4.01 34.78CspA-R2 Plant Analysis

Protocol: Three leaf stage rice seedlings (12 day old) were subjected toa cold stress of 10° C. for 3 days in presence of 1000 micromol./mt2/sec. and 70% RH in a growth chamber. After the stress treatmentthe plants were allowed to recover in the green house for 15 days and onthe 15^(th) day the growth observations were recorded. Each value is anaverage of 12 observations and the experiment was conducted by followingcompletely randomized (CRD) experimental design.

Results: Out of seven independent CspA transgenic lines tested 6 linesshowed improved cold tolerance compared to wild type. In this experimentplant height was reduced to close to 50% in cold treated control plants(WT) compared to non-stressed plants. Where as in transgenic plants withCspA gene reduction in plant height upon cold treatment varied 4.5% to22.50% among different independent lines (except one line wherereduction in growth was 47.09%). These results suggest that CspAimproves the cold tolerance of rice (Table 3).

TABLE 3 Three leaf stage cold stress recovery growth observations ofCspA R2 transgenic rice lines. Plant height at the Percent end ofrecovery (cm) reduction in plant height Lines Stressed Non-stressed overnon-stressed R2-362-3-1-2 28.75 ± 3.11 30.08 ± 2.9  4.5 R2-328-2-1-1 29.5 ± 2.92 35.58 ± 3.12 17.08 R2-362-7-1-2 15.83 ± 2.92 29.92 ± 1.7347.09 R2-365-4-5-3 26.08 ± 3.75 32.08 ± 2.27 18.7 R2-362-6-1-6 27.17 ±2.25 32.00 ± 1.76 15.05 R2-362-3-1-10 29.58 ± 3.50 38.17 ± 2.59 22.50R2-362-7-1-2 24.58 ± 3.42 27.25 ± 2.01 9.79 WT - 20.58 ± 1.73 37.92 ±8.59 46.05 NipponbareCspA-R3 Plant Analysis

Experiment I

Protocol: Three leaf stage seedlings were exposed to cold stress of 10degree Celsius for 3 days in presence of 1000 micro mol. of light. Laterthe seedlings were allowed to recover at 28 degree Celsius in thegreenhouse for 30 days and at the end of recovery the plant height andpercent seedling survival were recorded. (In this experiment 8replications were used for each transgenic line and 10 replications wereused for wild type.)

Results: The six transgenic lines subjected to cold stress performedbetter under cold stress than wild type. These results further confirmedthe R2 analysis data by showing improved cold tolerance (Table 4).

TABLE 4 Three leaf stage cold stress recovery growth observations ofCspA R3 transgenic rice lines. Percent Stressed- Non-stressed reductionin plant plant height (cm) at plant height (cm) at the height over non-Percent Lines the end of recovery end of recovery stress seedlingSurvival R3-362-3-1-2-2  25.5 ± 4.46 32.25 ± 5.03 20.93 100R3-362-3-1-3-2 25.62 ± 3.36 34.43 ± 6.24 25.58 66 R3-365-10-1-2-3 27.35± 3.24 33.75 ± 4.58 18.96 100 R3-362-6-1-2-1   28 ± 2.45 34.45 ± 2.2918.72 100 R3-362-7-1-2-3  27.5 ± 24.17 29.94 ± 5.03 8.1 100R3-362-7-1-3-3 27.88 ± 4.22 31.92 ± 2.89 12.65 100 WT-Nipponbare 26.25 ±3.95 36.34 ± 4.06 27.76 40 Note: Plant height was recorded only forsurvived plants and their averages are given above.

Experiment II

Protocol: Three leaf stage seedlings were exposed to cold stress of 10degree Celsius for 1 day in presence of 1000 micro mol. of light. Laterthe seedlings were allowed to recover at 28 degree Celsius in the greenhouse for 30 days and at the end of recovery the plant height andpercent seedling survival were recorded.

Results: The five transgenic lines subjected to cold stress performedbetter under cold stress than wild type. These results further confirmedthe R2 analysis data by showing improved cold tolerance (Table 5).

TABLE 5 Three leaf stage cold stress recovery growth observations ofCspA R3 transgenic rice lines. Non-stressed Percent Stressed- plantheight reduction in plant plant height (cm) at (cm) at end height overnon- Lines end of recovery of recovery stress R3-362-3-1-2-2 32.76 ±3.49 32.25 ± 5.03 Nil R3-362-3-1-3-2 36.11 ± 2.04 34.43 ± 6.24 NilR3-365-10-1-2-3 35.85 ± 2.94 33.75 ± 4.58 Nil R3-362-6-1-2-1 21.54 ±5.84 34.45 ± 2.29 37.4  R3-362-7-1-2-3 32.55 ± 2.73 29.94 ± 5.03 NilR3-362-7-1-3-3 32.17 ± 3.27 31.92 ± 2.89 Nil WT-Nipponbare 31.92 ± 2.6636.34 ± 4.06 12.16

Heat Stress Response at Three Leaf Stage

Plant Material Preparation:

Germination: Seeds were sterilized by treating with 0.01 percentmercuric chloride for 3 minutes and washed thoroughly (˜ten times indeionized water) to remove the traces of mercuric chloride. Sterilizedseeds were allowed to imbibe by soaking in milique water for 3 hours.The imbibed seeds were germinated on a sterilized moist filter paper at30° C. temperature and 60% RH using a seed germinator (SerwellInstruments Inc.).

Establishment of three leaf stage seedlings: The three day oldgerminated seedlings were transferred to portrays (52.5 mm (length)×26mm (depth)×5.2 mm (diameter)) in the green house having light intensityof 800 micro mol./mt2/sec. and 60% RH. The seedlings were grown tillthree-leaf stage (Approximately for 12 days) in portrays containing redsoil. Fertilizer solution was sprayed to the seedlings once a week tillthe completion of the experiments (N-75 PPM, P-32 PPM, K-32 PPM, Zn-8PPM, Mo-2 PPM, Cu-0.04 PPM, B-0.4 PPM and Fe-3.00 PPM).

CspA-R2 Plant Analysis

Protocol: Three leaf stage rice seedlings (12 day old) were subjected tothe heat stress of 50° C. for 3 hours in presence of 70% RH. After thestress treatment the plants were allowed to recover in the green housefor 15 days and on the 15^(th) day the growth observations wererecorded. Each value is an average of 12 observations.

Results: Out of seven independent CspA transgenic lines tested 6 linesshowed improved heat tolerance compared to wild type. In this experimentplant height was reduced by more than 50% in heat-treated control plants(WT) compared to no stressed plants. Where as in transgenic plants withCspA gene reduction in plant height upon heat treatment varied from 9.5%to 35% among different independent lines. These results suggest thatCspA improves the heat tolerance of rice (Table 6).

TABLE 6 Three leaf stage plant heat stress recovery growth observationsof CspA R2 transgenic rice lines. Plant height at the Percent end ofrecovery (cm) reduction in plant height Lines Stressed Non-stressed overnon-stressed R2-362-3-1-2 26.67 ± 4.97 30.08 ± 2.9  11.33 R2-328-2-1-126.17 ± 3.49 35.58 ± 3.12 26.41 R2-362-7-1-2 25.17 ± 1.94 29.92 ± 1.7315.87 R2-365-4-5-3 20.83 ± 1.17 32.08 ± 2.27 35.06 R2-362-6-1-6 23.17 ±1.83 32.00 ± 1.76 27.59 R2-362-3-1-10 29.33 ± 5.01 38.17 ± 2.59 23.15R2-362-7-1-2 24.67 ± 2.8  27.25 ± 2.01 9.4 WT- 18.5 3.51 37.92 ± 8.5951.21 NipponbareCspB-R3 Plant Analysis

Protocol: Three-leaf stage seedlings were exposed to high temperaturestress of 53 degree Celsius for 2 hours and later the seedlings wereallowed to recover at 28 degree Celsius in the greenhouse for 15 daysand at the end of recovery the plant height was recorded.

Results: Out of eight transgenic lines tested seven lines performedbetter under heat stress tested compared to wild type. These resultssuggest that CspB improves heat tolerance of rice (Table 7).

TABLE 7 Three leaf stage plant heat stress recovery growth observationsof CspB R3 transgenic rice lines. Non-stressed Percent Stressed-plantplant reduction height (cm) at end height (cm) at in plant height Linesof recovery end of recovery over non-stress R3-226-6-9-3 34.53 ± 2.1435.54 ± 2.07  2.84 R3-226-29-1-3-4 32.38 ± 1.47 37.06 ± 2.92  12.62R3-230-4-4-2-1 28.78 ± 4.16 35.06 ± 2.07  17.41 R3-230-34-1-2-1  33.3 ±3.94 37.6 ± 3.05 11.43 R3-238-1-1-3-4 33.96 ± 2.06 41.2 ± 3.83 17.57R3-257-3-1-3-1 33.76 ± 3.74 35.4 ± 2.07 4.63 R3-257-15-1-1-2 25.68 ±4.27 38.2 ± 3.11 32.77 R3-257-20-2-1-1 34.78 ± 1.7  42.2 ± 2.97 17.5WT-Taipei  25.5 ± 2.97 36.65 ± 4.8  30.42CspA-R3 Plant Analysis

Experiment I

Protocol: Three-leaf stage seedlings were exposed to high temperaturestress of 53 degree Celsius for 3 hours and later the seedlings wereallowed to recover at 28 degree Celsius in the greenhouse for 30 daysand at the end of recovery the plant height was recorded.

Results: These results confirmed the R2 analysis data by showingimproved heat tolerance (Table 8).

TABLE 8 Three leaf stage plant heat stress recovery growth observationsof CspA R3 transgenic rice lines. Non-stressed Percent Stressed - plantheight reduction in plant plant height (cm) at (cm) at end height overnon- Lines end of recovery of recovery stress R3-362-3-1-2-2 31.07 ±7.01 32.25 ± 5.03 3.6 R3-362-3-1-3-2 30.28 ± 4.74 34.43 ± 6.24 12.05R3-365-10-1-2-3 24.23 ± 7.60 33.75 ± 4.58 28.20 R3-362-6-1-2-1 26.93 ±2.97 34.45 ± 2.29 21.82 R3-362-7-1-2-3 29.52 ± 2.61 29.94 ± 5.03 1.40R3-362-7-1-3-3 21.30 ± 6.37 31.92 ± 2.89 33.27 WT-Nipponbare 22.68 ±2.96 36.34 ± 4.06 37.58

Experiment II

Protocol: Three leaf stage seedlings were exposed to high temperaturestress of 50 degree Celsius for 1 hour in the presence of 1000 micromol. of light and later the seedlings were allowed to recover at 28degree Celsius in the greenhouse for 30 days and at the end of recoverythe plant height was recorded.

Results: These results confirmed the R2 analysis data by showingimproved heat tolerance (Table 9).

TABLE 9 Three leaf stage plant heat stress recovery growth observationsof CspA R3 transgenic rice lines. Non-stressed Percent Stressed - plantheight reduction in plant plant height (cm) at (cm) at end height overnon- Lines end of recovery of recovery stress R3-362-3-1-2-2 31.57 ±2.39 32.25 ± 5.03 2.10 R3-362-3-1-3-2 34.20 ± 3.87 34.43 ± 6.24 0.6R3-365-10-1-2-3 31.63 ± 4.32 33.75 ± 4.58 6.28 R3-362-6-1-2-1 19.72 ±6.76 34.45 ± 2.29 42.75 R3-362-7-1-2-3 32.18 ± 3.25 29.94 ± 5.03 NilR3-362-7-1-3-3 32.80 ± 1.51 31.92 ± 2.89 Nil WT-Nipponbare 28.20 ± 2.7936.34 ± 4.06 22.39

Water Stress Response

Plant Material Preparation:

Germination: Seeds were sterilized by treating with 0.01 percentmercuric chloride for 3 min later washed thoroughly for ten times inmilique water to remove the traces of mercuric chloride. Sterilizedseeds were allowed to imbibe by soaking in milique water for 3 hours.The imbibed seeds were germinated on a sterilized moist filter paper at30° C. temperature and 60% RH using a seed germinator (SerwellInstruments Inc.).

CspB-R2 Plant Analysis

Experimental Protocol

The germinated seedlings (3 day old) were transferred to two differentlevels of water stress, created in PVC pots containing vermiculite,which is measured in terms of field capacity (FC). The FC—100% is asaturated condition (i.e. 100 g vermiculite requires 350 ml of water)(Sharp et. al., 1988, Plant physiol. 87: 50-57). The different levels ofwater stress (i.e. 50% FC and 25% FC) were created in a PVC potscontaining vermiculite by adding required amount of water. The waterstatus in different stress levels was constantly maintained, by addingeach day the amount of water lost due to evapotranspiration, through outthe experiment. The seedlings were allowed to grow for 15 days in thewater stress condition in the greenhouse in presence of 800 micromol./mt2/sec. light intensity and 60% RH. At 15^(th) day the growth ofroot and shoot were recorded and photographs were taken. Each treatmenthad 10 replications per line and they were completely randomized.

The percent reduction in growth was computed by adopting followingformula.

${\%\mspace{14mu}{reduction}\mspace{14mu}{over}\mspace{14mu}{absolute}\mspace{14mu}{control}} = {\frac{\frac{{Growth}\mspace{14mu}{of}\mspace{14mu}{root}}{{shoot}\mspace{14mu}{of}\mspace{14mu}{absolute}\mspace{14mu}{control}} - \frac{{Growth}\mspace{14mu}{of}\mspace{14mu}{root}}{{shoot}\mspace{14mu}{of}\mspace{14mu}{FC}\text{-}25\%}}{\frac{{Growth}\mspace{14mu}{of}\mspace{14mu}{root}}{{shoot}\mspace{14mu}{of}\mspace{14mu}{absolute}\mspace{14mu}{control}}} \times 100}$

Results: Four different CspB transgenic lines were analyzed for waterstress tolerance. All the CspB transgenic lines tested exhibitedsignificantly higher growth during stress compared to the wild typeplants. The transgenic lines including R2-257-15-1-1, R2-238-1-1-3,R2-257-3-1-6 and R2-226-6-9-3 exhibited least percent reduction in rootand shoot growth over non-stress control (FC—100%). The reduction inroot and shoot growth in these lines ranged between 11 to 25%. Where as,the wild type plants exhibited maximum reduction in growth, which isclose to 50%. These results suggest that CspA improves the water stresstolerance of rice (Table-10 and Table-11).

TABLE 10 Comparison of root and shoot growth at the end of water stressof cspB transgenic lines and the wild type. FC-100% FC-50% FC-25% LinesRoot Shoot R:S Root Shoot R:S Root Shoot R:S R2-257-15-1-1 9.2 ± 2.224.3 ± 2   .37   9 ± 1.27 23.7 ± 1.5  .37  8.1 ± 1.5 18.5 ± 2.2 .43R2-238-1-1-3 9.65 ± 2.7  26.3 ± 13.8 .36 8.35 ± 1.56 22.9 ± 1.26 .367.15 ± 1.0 18.1 ± 1.6 .39 R2-257-3-1-6 7.35 ± 2.2  26.1 ± 1.31 .28  6.4± 1.15 23.0 ± 1.57 .27  6.9 ± 1.07  19.5 ± 1.96 .35 R2-226-6-9-3 8.95 ±1.82 25.05 ± 1.6  .35 7.4 ± 1.2 19.0 ± 2.24 .38 7.25 ± 1.5  17.4 ± 2.15.41 WT - Taipei  9.2 ± 1.62 24.6 ± 1.58 .37  7.4 ± 1.66 22.4 ± 0.97 .336.58 ± 0.9 12.8 ± 3.2 .51 (Index: WT = wild type, R:S = Root to Shootratio)

TABLE 11 Comparison of percent reduction in growth of root and shoot ofcspB transgenic lines and the wild type. % Reduction in % Reduction in %Reduction in Lines root growth shoot growth root and shoot growthR2-257-15-1-1 11 23.8 20 R2-238-1-1-3 25 31 30 R2-257-3-1-6 6 25.2 21R2-226-6-9-3 19 30.5 27.5 WT-Taipei 28.4 47.9 42.8

CspA-R2 Plant Analysis

a. Plant Material Preparation:

Germination: Seeds were sterilized by treating with 0.01 percentmercuric chloride for 3 minutes and washed thoroughly for ten times inmilique water to remove the traces of mercuric chloride. Sterilizedseeds were allowed to imbibe by soaking in milique water for 3 hours.The imbibed seeds were germinated on a sterilized moist filter paper at30° C. temperature and 60% RH using a seed germinator (SerwellInstruments Inc.).

Establishment of three leaf stage seedlings: The three day oldgerminated seedlings were transferred to portrays (52.5 mm (length)×26mm (depth)×5.2 mm (diameter)) in the green house having light intensityof 800 micro mol./mt2/sec. and 60% RH. The seedlings were grown tillthree-leaf stage (Approximately for 12 days) in portrays containing redsandy loam soil. Fertilizer solution was sprayed to the seedlings once aweek till the completion of the experiments (N-75 PPM, P-32 PPM, K-32PPM, Zn-8 PPM, Mo-2 PPM, Cu-0.04 PPM, B-0.4 PPM and Fe-3.00 PPM).

Protocol: One-month-old seedlings were subjected to water stress forthree days in presence of 800 micro mol./mt2/sec. light and 60% RH inthe greenhouse. Water stress was imposed by withholding irrigation. Atthe end of three days, plants started showing the wilting symptom. Thestress was alleviated by irrigating the plants with water and 24 hourslater the observations on percent plants showing wilting symptoms wererecorded. A minimum of 12 plants was maintained per line per treatment.

Results: Out of seven independent CspA transgenic lines tested 6 linesshowed improved water stress tolerance compared to wild type. Sixty sixpercent of control plants did not recover from wilting after irrigationwhere as in CspA transgenic plants percentage of plants showing wiltingsymptoms after irrigation varied from 5% to 43% among differentindependent lines (except one line where percentage of plants showingwilting was 85%). These results suggest that CspA improves the waterstress tolerance in rice (Table 12).

TABLE 12 Water stress response of CspA R2 transgenic rice lines.Percentage Lines plants showing wilting R2-362-3-1-2 17 R2-328-2-1-1 43R2-362-7-1-2 85 R2-365-4-5-3 5 R2-362-6-1-6 Nil R2-362-3-1-10 15R2-362-7-1-2 8 WT- 66 Nipponbare

Salt Stress Response

CspB-R3 Plant Analysis

Protocol: Germinated seedlings (48 h. old) were subjected to salinitystress by transferring them to PVC pots with vermiculite containing 200mM of NaCl and grown for 10 days. After 10 days of stress the seedlingswere allowed to recover for 15 days by transferring them to a freshtrays of vermiculite containing water. The growth observation such asplant height was recorded at the end of recovery. This experiment wasconducted in the greenhouse by following Completely Randomized Design(CRD) and maintained eight replications per treatment.

Results: Seven CspB transgenic lines and wild type plants were subjectedto 200 mM NaCl stress. Under this condition five transgenic linesperformed better compared to wild type. These results suggest that CspBimproves tolerance of rice plants to salt stress (Table 13).

TABLE 13 Salt stress recovery growth observations of CspA R2 transgenicrice lines. Non-stressed Percent Stressed-plant plant height reductionin height (cm) at (cm) at end plant height Lines end of recovery ofrecovery over non-stressed R3-226-6-9-3 12.68 ± 2.83 23.48 ± 3.85 45.99R3-226-29-1-3-4 19.24 ± 3.46 25.54 ± 3.64 24.66 R3-230-4-4-2-1 15.39 ±3.05  25.2 ± 2.14 38.92 R3-230-34-1-2-1 15.78 ± 3.31 23.26 ± 1.98 32.15R3-238-1-1-3-4 13.41 ± 2.73 23.63 ± 4.61 43.25 R3-257-3-1-3-1 21.07 ±3.28 28.95 ± 4.37 27.64 R3-257-20-2-1-1 19.01 ± 3.98 26.35 ± 2.84 27.85WT-Taipei 14.71 ± 2.28 27.43 ± 2.75 46.37

R3 Water Stress Assay

Germinated seedlings (3 day old) from four independent transgenic lines(1,2,3,4) of cspA and wild type (Nipponbare—Number. 5) were subjected towater stress by transferring them into a pot containing vermiculite.Three levels of water regimes were maintained, they are 100% fieldcapacity (FC-100=3.72 ml of water/g vermiculite)

25% field capacity (FC25=0.93 ml of water/g vermiculite) 15% fieldcapacity (FC15=0.558 ml/g vermiculite). The seedlings were grown indifferent water regimes for 30 days in presence of 800 micromol./mt2/sec. light intensity and 60% RH in the greenhouse. The waterstatus in different stress levels was constantly maintained, by addingeach day the amount of water lost due to vapotranspiration, throughoutthe experiment. At the end of 30th day plants were allowed to recover byadding water to bring it the level of FC 100 and maintained for 15 days.During the experiment the growth observations such as plant height (pl.ht.) at the end of stress (ES) and root (R) shoot (S) length and dryweight at the end of the recovery were recorded.

Each treatment had 10 replications per line and they were completelyrandomized.

TABLE 14 Average sheet and root length (cm) at the end of recovery Linecode Lines FC100_Root FC100_Shoot FC25_Root FC25_Shoot FC15_RootFC15_Shoot 1 R2-362-3-1-3-4 24.5 ± 1.9 49.1 ± 3.6 17.0 ± 2.6 34.5 ± 2.412.5 ± 1.4 31.2 ± 1.8 2 R2-362-6-1-2-2 23.3 ± 1.3 45.6 ± 1.5 17.5 ± 1.831.5 ± 1.5 17.6 ± 2.3 32.0 ± 0.7 3 R2-362-7-1-3-3 24.5 ± 1.3 47.6 ± 3.317.8 ± 2.0 33.5 ± 1.7 15.9 ± 1.7 31.9 ± 1.7 4 R2-365-10-1-2-1 24.03 ±1.5  44.4 ± 2.2 13.8 ± 1.3 30.24 ± 1.1  13.9 ± 1.3 28.9 ± 0.9 5WT-Nipponbare 23.84 ± 1.25 44.8 ± 2.0 12.9 ± 1.8 31.6 ± 1.2 13.9 ± 1.931.5 ± 1.2

TABLE 15 Average shoot and root dry weight (mg) at the end of recoveryLine code Lines FC100_Root FC100_Shoot FC25_Root FC25_Shoot FC15_RootFC15_Shoot 1 R2-362-3-1-3-4 231 ± 21.8 563.9 ± 60.7  57.6 ± 6.8  189 ±16.3 44.7 ± 6.3 152.9 ± 22.1 2 R2-362-6-1-2-2 226 ± 14.2 531.8 ± 63   72.1 ± 5.1 179.8 ± 17  53.9 ± 7.9 146.3 ± 21.1 3 R2-362-7-1-3-3 229.5 ±30.2  533 ± 48.5  66.1 ± 11.9  183 ± 13.9 60.6 ± 5.7 147.4 ± 14.7 4R2-365-10-1-2-1 219 ± 43.5 557 ± 71.9 56.2 ± 9.3 173.9 ± 27.3 47.3 ± 3.1133.7 ± 7.7  5 WT-Nipponbare 226 ± 34.5 525 ± 31.3 61.1 ± 4.2 151.1 ±16.8 45.2 ± 7.5  132.2 ± 11.03

TABLE 16 Average shoot length (cm) at the end of stress Line code LinesFC100 FC25 FC15 1 R2-362-3-1-3-4   42 ± 4.6 28.4 ± 1.7 27.4 ± 2.1 2R2-362-6-1-2-2 40.4 ± 2.1 26.1 ± 1.1 25.2 ± 2.2 3 R2-362-7-1-3-3 40.1 ±2.7   27 ± 2.0 26.3 ± 1.4 4 R2-365-10-1-2-1 38.9 ± 2.3 26.3 ± 1.6 23.3 ±2.4 5 WT-Nipponbare  39.5 ± 1.05 24.2 ± 2.0 24.7 ± 1.9

Example 10 cspA

Construction of pMON73607 (FIG. 10)

1. Vector pMON61322 cut with NcoI and ApaI to open up backbone and dropout Csp A gene. Backbone fragment isolated by gel purification.

2. E. coli cspA gene PCR amplified from pMON56609 (FIG. 8) vector. PCRprimers used left the NcoI site at the 5′ end of the gene and created aSwaI and an ApaI site at the 3′ end.

3. Ligated PCR fragment and pMON61322 (FIG. 11) backbone. Transformedinto library efficiency DH5a cells. Screened colonies using ApaI andNcoI to identify clones with inserts.

4. Sequenced vector to confirm fidelity of the cspA gene and otherselected regions of the plasmid.

cspB

Construction of pMON73608 (FIG. 12)

1. Vector pMON61322 cut with NcoI and ApaI to open up backbone and dropout HVA1 gene. Backbone fragment isolated by gel purification.

2. Bacillus subtilis cspB gene PCR amplified from pMON56610 vector. PCRprimers used left the NcoI site at the 5′ end of the gene and created aSwaI and an ApaI site at the 3′ end.

3. Ligated PCR fragment and pMON61322 backbone. Transformed into libraryefficiency DH5a cells. Screened colonies using ApaI and NcoI to identifyclones with inserts.

4. Sequenced vector to confirm CspB gene and other selected regions ofthe plasmid.

Example 11 Maize Plant Transformation

Maize plants can be transformed by methods known in the art, forexample, see Examples 20-25 herein.

Example 12

Analysis of transgenic plants for copy number will be done in thefollowing manner.

Leaf tissue is collected from a young leaf; from as close to the base aspossible and from one side of the leaf. Samples are placed in 96-wellplates lyophilized overnight. Tissues are homogenized by placing three 3mm metal balls in each well and shaking using a Mega Grinder at 1200 rpmfor 2 minutes. DNA is extracted using standard buffers containingbeta-mercaptoethanol, Tris buffered to pH 8, EDTA, NaCl, and sodiumdodecyl sulfate. Extraction is performed with potassium acetate followedby chloroform and precipitation is performed with isopropanol. Followingcentrifugation, washing with ethanol solution, and drying, DNA isresuspended in Tris-EDTA buffer prior to further analysis.

DNA is digested with multiple restriction endonucleases and fragmentsare separated by non-denaturing agarose gel electrophoresis. DNA isdenatured by NaOH solution. The gel is neutralized in NaCl-containingTris buffer and blotted to nylon filters by capillary action. Nylonfilters are pre-hybridized in buffered solution containing salmon spermDNA prior to addition of appropriate probes, either radioactive orDIG-labeled. Following hybridization, blots are washed and detected byexposure to autoradiography film or detection of DIG with anti-DIGantibody conjugates and appropriate substrates.

Example 13

We are using the full length open reading frame of cspA and cspB forexpression in E. coli using vectors (Novagen, an affiliate of MerckKgaA, Darmstadt, Germany) that allow synthesis and purification ofHis-tagged antigen. Purified antigen will be used to generate polyclonalantibodies using a commercial provider, for example StrategicBiosolutions. Antibodies produced will be used to test plants forexpression of CSP proteins.

Example 14

Transgenic maize line advancement. Primary transformants are generatedin germplasm such as CORN OF GERMPLASM A, CORN OF GERMPLASM C, and CORNOF GERMPLASM D. Primary transformants are selfed as well as backcrossedto non-transgenic plants of the same inbred genotype. Seed from selfedplants is planted in the field and assayed by Taqman zygosity assay toidentify putative homozygous selections, putative heterozygousselections, and negative selections. Putative heterozygous selectionsare crossed with multiple plants of appropriate testers, e.g. CORN OFGERMPLASM B and CORN OF GERMPLASM D. Hybrid seed is harvested, handshelled, and pooled by selection. Other breeding methods may also beemployed, for example, see example 29 herein.

Example 15

Seedlings will receive a treatment that limits available water to asub-optimal level such that the treatment results in a measurablephenotypic response. For example, this treatment could take the form ofrestricting the amount of water over a number of days leading to aprogressive water deficit, or the form of an acute deficit byosmotically stressing the seedlings hydroponically or with a salttreatment. Transgene positive plants will be screened for an improvedphenotypic response to the treatment. The phenotypic responses measuredmay include shoot growth rate or dry weight accumulation during thetreatment or following a post-treatment recovery period, wilting or wiltrecovery, and root growth rates and dry weight accumulation. Those withimproved response will be advanced to a field efficacy trial. Screenswill require a number of transgene positive and transgene negativeplants to be grown in small pots in a controlled environment such as agrowth chamber or greenhouse. The number of plants screened is dictatedby the variance associated with treatments applied and phenotypesmeasured.

Example 16

Field grown plants will receive a treatment that limits available waterto a sub-optimal level such that the treatment results in a measurablephenotypic response. For example, this treatment could take the form ofrestricting the amount of water available to the plants over a number ofdays leading to a progressive water deficit either during latevegetative or early reproductive development of the plants. Transgenepositive plants will be screened for an improved phenotypic response tothe treatment relative to transgene negative plants. The phenotypicresponses measured may include shoot growth rate during the treatment,leaf wilting, grain yield, and ear yield components such as kernelnumber and kernel weight. Those events with improved response will beadvanced to a first year yield trial. Screens will be applied at typicalplanting densities at two dryland field locations with controllableirrigation. The number of plants screened is dictated by the varianceassociated with treatments applied and phenotypes measured.

Example 17

Several of the genes described will be cloned, transformed into plants,and be phenotyped in a manner similar to the following (Examples 17-30).For example, nucleotides and nucleotides encoding SEQ ID NOS: 4-53.

Construction of the Destination Vector.

A GATEWAY™ Destination (Invitrogen Life Technologies, Carlsbad, Calif.)plant expression vector was constructed (pMON65154, FIG. 13) usingmethods known to those of skill in the art. The elements of theexpression vector are summarized in Table 17. The backbone of theplasmid pMON65154 comprising the bacterial replication functions and anampicillin resistance gene expressed in E. coli were derived from theplasmid pSK-. The plant expression elements in pMON64154 are availableto those of skill in the art and references are provided for eachelement in Table 17. All references in Table 17 to location refer tobase pair coordinates for each element on the plasmid map disclosed inFIG. 13. Generally, pMON65154 comprises a selectable marker expressioncassette comprising a Cauliflower Mosaic Virus 35S promoter operablylinked to a gene encoding neomycin phosphotransferase II (nptII). The 3′region of the selectable marker expression cassette comprises the 3′region of the Agrobacterium tumefaciens nopaline synthase gene (nos)followed 3′ by the 3′ region of the potato proteinase inhibitor II(pinII) gene. The plasmid pMON 65154 further comprises a plantexpression cassette into which a gene of interest may be inserted usingGATEWAY™ cloning methods. The GATEWAY™ cloning cassette is flanked 5′ bya rice actin 1 promoter, exon and intron and flanked 3′ by the 3′ regionof the potato pinII gene. Using GATEWAY™ methods, the cloning cassettewas replaced by a gene of interest. The vector pMON65154 and derivativesthereof comprising a gene of interest, were particularly useful inmethods of plant transformation via direct DNA delivery, such asmicroprojectile bombardment. One of skill in the art could construct anexpression vector with similar features using methods known in the art.Furthermore, one of skill in the art would appreciate that otherpromoters and 3′ regions would be useful for expression of a gene ofinterest and other selectable markers may be used.

TABLE 17 Elements of Plasmid pMON65154 CASSETTE FUNCTION ELEMENTLOCATION REFERENCE Plant gene of Promoter Rice actin 1 1796-2638 Wang etal., 1992 interest expression Enhancer Rice actin 1 exon 2639-3170 Wanget al., 1992 1, intron 1 GATEWAY ™ Recombination AttR1 3188-3312GATEWAY ™ Cloning cloning Technology Instruction Manual (Invitrogen LifeTechnologies, Carlsbad, CA) Bacterial CmR gene 3421-4080GATEWAY ™ Cloning chloramphenical Technology Instruction resistance geneManual (Invitrogen Life Technologies, Carlsbad, CA) Bacterial negativeccdA, ccdB 4200-4727 GATEWAY ™ Cloning selectable markers genesTechnology Instruction Manual (Invitrogen Life Technologies, Carlsbad,CA) GATEWAY ™ attR2 4768-4892 GATEWAY ™ Cloning recombination siteTechnology Instruction Manual (Invitrogen Life Technologies, Carlsbad,CA) Plant gene of 3′ region Potato pinII 4907-5846 An et al., 1989interest expression cassette Plant selectable Promoter Cauliflower5895-6218 US Patent # 5352605 marker gene Mosaic Virus expressioncassette 35S Selectable marker nptII 6252-7046 US Patent # 6174724 gene3′ region nos 7072-7327 Bevan et al., 1983 3′ region pinII 7339-8085 Anet al., 1989 Maintenance in E. coli Origin of replication ColE1 858-1267 Oka et al, 1979 Maintenance in E. coli Origin of replicationF1 8273-3673 Ravetch et al., 1977 Maintenance in E. coli Ampicillinresistance bla 8909-551  Heffron et al., 1979

A separate plasmid vector (pMON72472, FIG. 14) was constructed for usein Agrobacterium mediated methods of plant transformation. The plasmidpRG76 comprises the gene of interest plant expression, GATEWAY™ cloning,and plant selectable marker expression cassettes present in pMON65154.In addition left and right T-DNA border sequences from Agrobacteriumwere added to the plasmid. The right border sequence is located 5′ tothe rice actin 1 promoter and the left border sequence is located 3′ tothe pinII 3′ sequence situated 3′ to the nptII gene. Furthermore thepSK-backbone of pMON65164 was replaced by a plasmid backbone tofacilitate replication of the plasmid in both E. coli and Agrobacteriumtumefaciens. The backbone comprises an oriV wide host range origin ofDNA replication functional in Agrobacterium, the rop sequence, a pBR322origin of DNA replication functional in E. coli and aspectinomycin/streptomycin resistance gene for selection for thepresence of the plasmid in both E. coli and Agrobacterium.

The elements present in plasmid vector pRG81 are described in Table 18.

TABLE 18 Genetic Elements of Plasmid Vector pRG81 CASSETTE FUNCTIONELEMENT LOCATION REFERENCE Plant gene of Promoter Rice actin 1 5610-6452Wang et al., 1992 interest expression Enhancer Rice actin 1 6453-6984Wang et al., 1992 exon 1, intron 1 GATEWAY ™ Recombination AttR17002-7126 GATEWAY ™ Cloning cloning Technology Instruction Manual(Invitrogen Life Technologies, Carlsbad, CA) Bacterial CmR gene7235-7894 GATEWAY ™ Cloning chloramphenical Technology Instructionresistance gene Manual (Invitrogen Life Technologies, Carlsbad, CA)Bacterial negative ccdA, ccdB 8014-8541 GATEWAY ™ Cloning selectablemarkers genes Technology Instruction Manual (Invitrogen LifeTechnologies, Carlsbad, CA) GATEWAY ™ attR2 8582-8706 GATEWAY ™ Cloningrecombination site Technology Instruction Manual (Invitrogen LifeTechnologies, Carlsbad, CA) Plant gene of 3′ region Potato pinII8721-9660 An et al., 1989 interest expression cassette Plant selectablePromoter Cauliflower  1-324 US Patent # 5352605 marker gene Mosaic Virusexpression 35S cassette Selectable marker nptII  358-1152 US Patent #6174724 gene 3′ region nos 1178-1433 Bevan et al., 1983 3′ region pinII1445-2191 An et al., 1989 Agrobacterium DNA transfer Left border2493-2516 Zambryski et al., 1982; mediated GenBank Accessiontransformation AJ237588 Maintenance of Origin of replication Ori-V2755-3147 Honda et al., 1988 plasmid in E. coli or AgrobacteriumMaintenance of Origin of replication ColE1 3545-4199 Oka et al., 1972plasmid in E. coli Maintenance of Spectinomycin/ststreptomycin Spc/Str4242-5030 Fling et al., 1985 plasmid in E. coli or resistanceAgrobacterium Agrobacterium DNA transfer Right 5514-5538 Zambryski etal., 1982; mediated border GenBank Accession transformation AJ237588

Example 18

Coding sequences were amplified by PCR prior to insertion in a GATEWAY™Destination plant expression vector such as pMON65154 (FIG. 13). Allcoding sequences were available as either a cloned full length sequenceor as DNA sequence information which allowed amplification of thedesired sequence from a cDNA library. Primers for PCR amplification weredesigned at or near the start and stop codons of the coding sequence, inorder to eliminate most of the 5′ and 3′ untranslated regions. PCRproducts were tailed with attB1 and attB2 sequences in order to allowcloning by recombination into GATEWAY™ vectors (Invitrogen LifeTechnologies, Carlsbad, Calif.).

Two methods were used to produce attB flanked PCR amplified sequences ofinterest. Both methods are described in detail in the GATEWAY™ CloningTechnology Instruction Manual (Invitrogen Life Technologies, Carlsbad,Calif.). In the first method, a single primer set comprising attB andtemplate specific sequences was used. The primer sequences are asfollows:

attB1 forward primer: (SEQ ID NO: 71) 5′ GGG CAC TTT GTA CAA GAA AGC TGGGTN template specific sequence 3′ attB2 reverse primer (SEQ ID NO: 72)5′ GGGG CAC TTT GTA CAA GAA AGC TGG GTN template specific sequence 3′

Alternatively, attB adapter PCR was used to prepare attB flanked PCRproducts. attB1 adapter PCR uses two sets of primers, i.e., genespecific primers and primers to install the attB sequences. Desired DNAsequence primers were designed which included 12 base pairs of the attB1or attB2 sequences at the 5′ end. The primers that were used were asfollows:

attB1 gene specific forward primer (SEQ ID NO: 73) 5′ CCTGCAGGACCATGforward gene specific primer 3′ attB2 gene specific reverse primer (SEQID NO: 74) 5′ CCTGCAGGCTCGAGCTA reverse gene specific primer 3′

The second set of primers were attB adapter primers with the followingsequences:

attB1 adapter forward primer (SEQ ID NO: 75)5′ GGGGACAAGTTTGTACAAAAAAGCAGGCTCCTGCAGGACCATG 3′ attB2 adapter reverseprimer (SEQ ID NO: 76) 5′GGGGACCACTTTGTACAAGAAAGCTGGGTCCCTGCAGGCTCGAGCTA 3′

attB1 and attB2 flanked sequences were amplified by PCR according to themethods described by Invitrogen Life Technologies (Carlsbad, Calif.).attB flanked PCR products were purified and recovered from a gel asdescribed above.

In some instances, attB flanked sequences were recovered from PCR, butcould not be inserted into the Donor Vector using GATEWAY™ technology.Conventional cloning methods using ligases were used to insert a DNAsequence into an Entry Vector (Invitrogen Life Technologies, Carlsbad,Calif.) when GATEWAY™ recombination into the Donor Vector failed.

The choice of Entry Vector depended on the compatibility of restrictionendonuclease sites in the Entry Vector and desired insert sequence. TheEntry Vector was digested with a selected restriction endonuclease toremove the ccdB gene, dephosphorylated and gel purified. The selectedrestriction endonuclease depended on the Entry Vector used and thesequence of the desired insert sequence. For example, the ccdB gene wasremoved from pENTR11 (FIG. 15) using EcoR1 or other combinations ofrestriction endonucleases such as EcoRV, and XmaI or NcoI and XhoI.Other restriction nucleases could be used with other Entry Vectors foruse in the GATEWAY™ process. To use restriction endonuclease digestedEntry Vectors, it was necessary to be able to produce compatible stickyends on the desired PCR product. Sticky ends could be produced by anumber of methods known to those of skill in the art, such asrestriction endonuclease digestion, adapter ligation or addition ofrestriction sites during PCR.

In some instances, it was not possible to produce compatible sticky endson a PCR fragment and an Entry Vector. Alternatively, compatible stickyends could be produced directed by restriction enzyme digestion of acDNA clone. It was possible, however, to blunt end ligate PCR fragmentsinto an Entry Vector. Using this method, the Entry Vector was cut with arestriction endonuclease to remove the ccdB gene. A gel purified linearEntry Vector was made blunt ended with T4 DNA polymerase. One of skillin the art is aware of other methods of making blunt ended DNAmolecules, such as the use of Klenow DNA polymerase. The PCR product wasmade blunt ended and preferably dephosphorylated by incubation with T4DNA polymerase, or another suitable polymerase, T4 polynucleotide kinaseand a phosphatase enzyme. The Entry Vector and PCR product were bluntend ligated using methods known in the art. Ligation products weretransformed into E. coli and plasmids from individual colonies analyzedfor presence of the insert DNA and the desired orientation relative tothe attL sites in the Entry Vector. Clones with the attL1 sequence nextto the amino end of the open reading frame were selected.

Preferably, the TA method of cloning PCR products (Marchuk et al., 1991)was used when attB flanked PCR products could not be inserted into aplasmid using GATEWAY™ methods. The TA method takes advantage of Taqpolymerase terminal transferase activity. An Entry Vector was cut with arestriction endonuclease and made blunt ended using the methodsdescribed herein. The blunt ended linear Entry Vector was incubated withdTTP and Taq polymerase resulting in the addition of a single thymidineresidue at the 3′ end of each DNA strand. Since Taq polymerase has astrong preference for dATP, PCR products are most often produced with asingle adenosine added to the 3′ end. Therefore, the Entry Vector andPCR product have complimentary single base 3′ overhangs. Followingligation under conditions known to those of skill in the art, plasmidswere transformed into E. coli. Plasmids were isolated from individualcolonies and analyzed to identify plasmids with the desired insert inthe correct orientation. Alternatively, PCR products, tailed with attBsites were TA cloned into a commercial TA cloning vector, such as pGEM-TEASY (Promega Corporation, Madison, Wis.).

All PCR amplification products were sequenced prior to introduction intoa plant. PCR inserts in Destination expression vectors produced byGATEWAY™ methods were sequenced to confirm that the inserted sequencedencoded the expected amino acid sequence. If Entry Vectors were producedusing ligation methods, the inserted sequence was sequenced in the EntryVector prior to production of the Destination expression vector usingGATEWAY™ technology. Point mutations which did not affect the amino acidcoding sequence, i.e., silent mutations, were accepted.

Example 19 Construction of Expression Vectors

GATEWAY™ cloning methods (Invitrogen Life Technologies, Carlsbad,Calif.) were used to construct expression vectors for use in maizetransformation. The GATEWAY™ methods are fully described in the GATEWAY™Cloning Technology Instruction Manual (Invitrogen Life Technologies,Carlsbad, Calif.). Use of the GATEWAY™ system facilitates highthroughput cloning of coding sequences into a plant expression vector.Gene sequences flanked by attB1 and attB2 sequences were produced by PCRas described above. Depending on which recombination sequence, attB1 andattB2, was placed 5′ and 3′ to the coding sequence, sense or antisenseexpression vectors were produced. A plant expression vector, pMON65154(FIG. 13), into which any coding sequence could be inserted in a senseor antisense orientation was constructed as described in Example 1 andwas used as a destination vector in the GATEWAY™ cloning process.

Two alternative processes were used for inserting a PCR amplified codingsequence into a plant expression vector. In the first method, a PCRproduct comprising the coding sequence of interest flanked by attB1 andattB2 sequences at the 5′ and 3′ ends was incubated with the donorvector (pDONR201™, Invitrogen Life Technologies, Carlsbad, Calif.) inthe presence of BP CLONASE™. GATEWAY™ entry clones were produced fromthis reaction and transformed into E. coli. Plasmid DNA was isolatedfrom entry clones. Inserted coding sequences could be sequenced fromentry vectors in order to confirm the fidelity of PCR amplification.Plasmid DNA, isolated from entry clone E. coli colonies, was incubatedwith linearized destination vector, preferably pMON65154, in thepresence of LR CLONASE™ to produce plant expression vectors comprisingthe coding sequence of interest. DNA from the LR CLONASE™ reaction wastransformed into E. coli. Plasmid DNA from destination expressionvectors was isolated and sequenced in order to determine correctorientation and sequence of the plant expression vector.

In the second method of generating plant expression vectors, a PCRproduct flanked by attB1 and attB2 sequences was incubated with a donorvector (pDONR201™, Invitrogen Life Technologies, Carlsbad, Calif.), andBP CLONASE™ as described above. Following incubation, an aliquot of thereaction mix was further incubated with linearized destination vectorand LR CLONASE™. The resultant DNA was transformed into E. coli andplant expression vectors containing the coding sequence of interestselected using PCR or Southern blot analysis techniques known in theart. Both methods of producting plant expression vectors comprising acoding sequence of interest were described by Invitrogen LifeTechnologies (GATEWAY™ Cloning Technology Instruction Manual).

Alternatively, Entry Vectors were produced using restrictionendonucleases and ligases. Entry Vectors are available from InvitrogenLife Technologies (Carlsbad, Calif.). Each entry vector, e.g., pENTR1A,pENTR2B, pENTR3C, pENTR4, and pENTR11, has unique cloning and expressionfeatures. pENTR11 was preferably used in the practice of the presentinvention. Those of skill in the art will recognize the usefulness ofthe other Entry Vectors. Before using restriction endonucleases andligases to insert desired sequences into one of the Entry Vectors, itwas necessary to restriction digest the Entry Vector on each side of theccdB gene. A number of different combinations of restrictionendonucleases were used depending on the restriction sites present onthe DNA sequence to be inserted into the Entry Vector. Preferably theEntry Vector was dephosphorylated and gel purified after restrictiondigestion. The desired DNA sequence was inserted into the Entry Vectorusing conventional methods of molecular biology known to those of skillin the art. TA cloning (U.S. Pat. No. 5,827,657) is a preferable methodof cloning PCR fragments into an Entry Vector.

Vectors (designated as pMON and a 5 digit number) and coding sequencescontained therein that were produced using the GATEWAY™ cloning methodsare, for example, SEQ ID NOS: 4-28. It is expected that some of thecoding sequences of the present invention may be cloned into a plantexpression vectors using the methods described herein.

Example 20

CORN OF GERMPLASM A plants were grown in the greenhouse. Ears wereharvested from plants when the embryos were 1.5 to 2.0 mm in length,usually 10 to 15 days after pollination, and most frequently 11 to 12days after pollination. Ears were surface sterilized by spraying orsoaking the ears in 80% ethanol, followed by air drying. Alternatively,ears were surface sterilized by immersion in 50% CLOROX™ containing 10%SDS for 20 minutes, followed by three rinses with sterile water.

Immature embryos were isolated from individual kernels using methodsknown to those of skill in the art. Immature embryos were cultured onmedium 211 (N6 salts, 2% sucrose, 1 mg/L 2,4-D, 0.5 mg/L niacin, 1.0mg/L thiamine-HCl, 0.91 g/L L-asparagine, 100 mg/L myo-inositiol, 0.5g/L MES, 100 mg/L casein hydrolysate, 1.6 g/L MgCl₂, 0.69 g/L L-proline,2 g/L GELGRO™, pH 5.8) containing 16.9 mg/L AgNO₃, (designated medium211V) for 3-6 days, preferably 3-4 days prior to microprojectilebombardment.

Example 21

Methods of Agrobacterium mediated transformation of maize cells andother monocots are known (Hiei et al., 1997; U.S. Pat. No. 5,591,616;U.S. Pat. No. 5,981,840; published EP patent application EP 0 672 752).Although various strains of Agrobacterium may be used (see referencesabove), strain ABI is used preferably by the present inventors. The ABIstrain of Agrobacterium is derived from strain A208, a C58 nopaline typestrain, from which the Ti plasmid was eliminated by culture at 37° C.,and further containing the modified Ti plasmid pMP90RK (Koncz andSchell, 1986). An Agrobacterium tumefaciens binary vector system (An etal., 1998) is preferably used to transform maize. Alternativecointegrating Ti plasmid vectors have been described (Rogers et al.,1988) and could be used to transform maize. A binary vector comprisingone or more genes of interest may be introduced into a disarmedAgrobacterium strain using electroporation (Wen-jun and Forde, 1989) ortriparental mating (Ditta et al., 1980). A binary vector may contain aselectable marker gene, a screenable marker gene and/or one or moregenes that confer a desirable phenotypic trait on the transformed plant.An exemplary binary vector, pMON30113, is shown in FIG. 4. Other binaryvectors may be used and are known to those of skill in the art.

Prior to co-culture of maize cells, Agrobacterium cells may be grown at28° C. in LB (DIFCO) liquid medium comprising appropriate antibiotics toselect for maintenance of the modified Ti plasmid and binary vector. Forexample, ABI/pMON30113, may be grown in LB medium containing 50 ug/mlkanamycin to select for maintenance of the pMP90RK modified Ti plasmidand 100 ug/ml spectinomycin to select for maintenance of the binaryvector pMON30113. It will be obvious to one of skill in the art to useappropriate selection agents to maintain plasmids in the hostAgrobacterium strain. Prior to inoculation of maize cells, Agrobacteriumcells are grown overnight at room temperature in AB medium (Chilton etal., 1974) comprising appropriate antibiotics for plasmid maintenanceand 200 uM acetosyringone. Immediately prior to inoculation of maizecells, Agrobacterium are preferably pelleted by centrifugation, washedin ½ MSVI medium (2.2 g/L GIBCO (Carlsbad, Calif.) MS salts, 2 mg/Lglycine, 0.5 g/L niacin, 0.5 g/L L-pyridoxine-HCl, 0.1 mg/L thiamine,115 g/L L-proline, 10 g/L D-glucose, and 10 g/L sucrose, pH 5.4)containing 200 uM acetosyringone, and resuspended at 0.1 to 1.0×10⁹cells/ml in ½ MSPL medium (2.2 g/L GIBCO (Carlsbad, Calif.) MS salts, 2mg/L glycine, 0.5 g/L niacin, 0.5 g/L L-pyridoxine-HCl, 0.1 mg/Lthiamine, 115 g/L L-proline, 26 g/L D-glucose, 68.5 g/L sucrose, pH 5.4)containing 200 uM acetosyringone. One of skill in the art may substituteother media for ½ MSVI or ½ MSPL.

Immature maize embryos are isolated as described previously. Embryos areinoculated with Agrobacterium 0-7 days after excision, preferablyimmediately after excision. Alternatively, immature embryos may becultured for more than 7 days. For example, embryogenic callus may beinitiated as described above and co-cultured with Agrobacterium.Preferably, immature maize embryos are excised, immersed in anAgrobacterium suspension in ½ MSPL medium prepared as described aboveand incubated at room temperature with Agrobacterium for 5-20 minutes.

Following inoculation embryos are transferred to one-half strength MSmedium (Murashige and Skoog, 1962) containing 3.0 mg/L2,4-dichlorophenyoxyacetic acid (2,4-D), 1% D-glucose, 2% sucrose, 0.115g/L L-proline, 0.5 mg/L thiamine-HCl, 200 uM acetosyringone, and 20 uMsilver nitrate or silver thiosulfate. Immature embryos are co-culturedwith Agrobacterium for 1 to 3 days at 23° C. in the dark. One of skillin the art may substitute other media for the described media.

Co-cultured embryos are transferred to medium 15AA (462 mg/L (NH4)SO4,400 mg/L KH2PO4, 186 mg/L MgSO4-7H20, 166 mg/L CaCl2-2H20, 10 mg/LMnSO4-H20, 3 mg/L H3B03, 2 mg/L ZnSO4-7H20, 0.25 mg/L NaMoO4-2H20, 0.025mg/L CuSO4-5H20, 0.025 mg/L CoCl2-6H20, 0.75 mg/L KI, 2.83 g/L KNO3, 0.2mg/L niacin, 0.1 mg/L thiamine-HCl, 0.2 mg/L pyridoxine-HCl, 0.1 mg/LD-biotin, 0.1 mg/L choline chloride, 0.1 mg/L calcium pantothenate, 0.05mg/L folic acid, 0.05 mg/L p-aminobenzoic acid, 0.05 mg/L riboflavin,0.015 mg/L vitamin B12, 0.5 g/L casamino acids, 33.5 mg/L Na2EDTA, 1.38g/L L-proline, 20 g/L sucrose, 10 g/L D-glucose), or MS mediumcontaining 1.5 mg/L 2,4-D, 500 mg/L carbenicillin, 3% sucrose, 1.38 g/LL-proline and 20 uM silver nitrate or silver thiosulfate and culturedfor 0 to 8 days in the dark at 27° C. without selection. Culture mediaused for selection of transformants and regeneration of plantspreferably contains 500 mg/L carbenicillin. One of skill in the art maysubstitute other antibiotics that control growth of Agrobacterium. Otherculture media that support cell culture may be used alternatively. Inthe absence of a delay of selection (0 day culture), selection may beinitiated on 25 mg/L paromomycin. Selection medium may comprise medium211 (described above) or a variant of medium 211 in which N6 salts arereplaced by MS salts. After two weeks, embryogenic callus aretransferred to culture medium containing 100 mg/L paromomycin andsubcultured at about two week intervals. When selection is delayedfollowing co-culture, embryos are initially cultured on mediumcontaining 50 mg/L paromomycin followed by subsequent culture ofembryogenic callus on medium containing 100-200 mg/L paromomycin. One ofskill in the art will culture tissue on concentrations of paromomycinwhich inhibit growth of cells lacking the selectable marker gene, but aconcentration on which transformed callus will proliferate.Alternatively, one may use other selectable markers to identifytransformed cells. It is believed that initial culture on 25 to 50 mg/Lparomocyin for about two weeks, followed by culture on 50-200 mg/Lparomoycin will result in recovery of transformed callus. Transformantsare recovered 6 to 8 weeks after initiation of selection. Plants areregenerated from transformed embryogenic callus as described above fortransformants recovered following microprojectile bombardment.

Example 22 Agrobacterium Mediated Transformation of Maize Callus

This example describes methods for transformation of maize callus usingAgrobacterium. The method is exemplified using an nptII selectablemarker gene and paromomycin selective agent. One of skill in the artwill be aware of other selectable marker and selective agentcombinations that could be used alternatively.

Callus was initiated from immature embryos using methods known to thoseof skill in the art. For example, 1.5 mm to 2.0 mm immature embryos wereexcised from developing maize seed of a genotype such as CORN OFGERMPLASM A and cultured with the embryonic axis side down on medium211V, usually for 8-21 days after excision. Alternatively, establishedan established callus culture may be initiated and maintained by methodsknown to those of skill in the art.

Agrobacterium was prepared for inoculation of plant tissue according tothe methods described in Example 21. Fifty to 100 pieces of callus wastransferred to a 60 mm×20 mm petri dish containing about 15 ml ofAgrobacterium suspension at 0.1 to 1.0×10⁹ cfu/ml. A piece of callus wasusually all of the callus produced by an immature embryo in up to 21days of culture or a piece of established callus of 2 mm to 8 mm indiameter. Callus was incubated for about 30 minutes at room temperaturewith the Agrobacterium suspension, followed by removal of the liquid byaspiration.

About 50 μL of sterile distilled water was added to a Whatman #1 filterpaper in a 60 mm×20 mm petri dish. After 1-5 minutes, 15 to 20 pieces ofcallus were transferred to each filter paper and the plate sealed withPARAFILM®, for example. The callus and Agrobacterium were co-culturedfor about 3 days at 23° C. in the dark.

Calli were transferred from filter paper to medium 211 with 20 μM silvernitrate and 500 mg/L carbenicillin and cultured in the dark at 27° C. to28° C. for 2-5 days, preferably 3 days. Selection was initiated bytransferring callus to medium 211 containing 20 μM silver nitrate, 500mg/L carbenicillin and 25 mg/L paromomycin. After 2 weeks culture in thedark at 27° C. to 28° C., callus was transferred to medium 211 with 20μM silver nitrate, 500 mg/L carbenicillin and 50 mg/L paromomycin(medium 211QRG). Callus was subcultured after two weeks to fresh medium211 QRG and further cultured for two weeks in the dark at 27° C. to 28°C. Callus was then transferred to medium 211 with 20 μM silver nitrate,500 mg/L carbenicillin and 75 mg/L paromomycin. After 2-3 weeks culturein the dark at 27° C. to 28° C., paromomycin resistant callus wasidentified. One of skill in the art would recognize that times betweensubcultures of callus are approximate and one may be able to acceleratethe selection process by transferring tissue at more frequent intervals,e.g., weekly rather than biweekly.

Plants were regenerated from transformed callus, transferred to soil andgrown in the greenhouse as described. Following Agrobacterium mediatedtransformation, medium 217 (see Example 9) further contained 500 mg/Lcarbenicillin and medium 127T (see Example 9) further contained 250 mg/Lcarbenicillin. Transformed maize plants comprising genes of the presentinvention that were produced using Agrobacterium mediated transformationare summarized in table Y.

Example 23 Methods of Microprojectile Bombardment

Approximately four hours prior to microprojectile bombardment, immatureembryos were transferred to medium 211 SV (medium 211V with the additionof sucrose to 12%). Twenty five immature embryos were preferably placedin a 60×15 mm petri dish, arranged in a 5×5 grid with the coleoptilarend of the scutellum pressed slightly into the culture medium at a 20degree angle. Tissue was maintained in the dark prior to bombardment.

Prior to microprojectile bombardment, a suspension of gold particles wasprepared onto which the desired DNA was precipitated. Ten milligrams of0.6 μm gold particles (BioRad) were suspended in 50 μL buffer (150 mMNaCl, 10 mM Tris-HCl, pH 8.0). Twenty five μL of a 2.4 nM solution ofthe desired DNA was added to the suspension of gold particles and gentlyvortexed for about five seconds. Seventy five μL, of 0.1M spermidine wasadded and the solution vortexed gently for about 5 seconds. Seventy fiveμL of a 25% solution of polyethylene glycol (3000-4000 molecular weight,American Type Culture Collection) was added and the solution was gentlyvortexed for five seconds. Seventy five μL of 2.5 M CaCl₂ was added andthe solution vortexed for five seconds. Following the addition of CaCl₂,the solution was incubated at room temperature for 10 to 15 minutes. Thesuspension was subsequently centrifuged for 20 seconds at 12,000 rpm(Sorval MC-12V centrifuge) and the supernatant discarded. The goldparticle/DNA pellet was washed twice with 100% ethanol and resuspendedin 10 mL 100% ethanol. The gold particle/DNA preparation was stored at−20° C. for up to two weeks.

DNA was introduced into maize cells using the electric dischargeparticle acceleration gene delivery device (U.S. Pat. No. 5,015,580).The gold particle/DNA suspension was coated on Mylar sheets (Du PontMylar polyester film type SMMC2, aluminum coated on one side, overcoated with PVDC co-polymer on both sides, cut to 18 mm square) bydispersion of 310 to 320 of the gold particle/DNA suspension on a sheet.After the gold particle suspension settled for one to three minutes,excess ethanol was removed and the sheets were air dried.Microprojectile bombardment of maize tissue was conducted as describedin U.S. Pat. No. 5,015,580. AC voltage may be varied in the electricdischarge particle delivery device. For microprojectile bombardment ofCORN OF GERMPLASM A pre-cultured immature embryos, 35% to 45% of maximumvoltage was preferably used. Following microprojectile bombardment,tissue was cultured in the dark at 27° C.

Example 24 Selection of Transformed Cells

Transformants were selected on culture medium comprising paromomycin,based on expression of a transgenic neomycin phosphotransferase II(nptII) gene. Twenty four hours after DNA delivery, tissue wastransferred to 211V medium containing 25 mg/L paromomycin (medium211HV). After three weeks incubation in the dark at 27° C., tissue wastransferred to medium 211 containing 50 mg/L paromomycin (medium 211G).Tissue was transferred to medium 211 containing 75 mg/L paromomycin(medium 211XX) after three weeks. Transformants were isolated following9 weeks of selection. Table Y discloses results of transformantexperiments using the methods of microprojectile bombardment disclosedherein.

Example 25 Regeneration of Fertile Transgenic Plants

Fertile transgenic plants were produced from transformed maize cells.Transformed callus was transferred to medium 217 (N6 salts, 1 mg/Lthiamine-HCl, 0.5 mg/L niacin, 3.52 mg/L benzylaminopurine, 0.91 mg/LL-asparagine monohydrate, 100 mg/L myo-inositol, 0.5 g/L MES, 1.6 g/LMgCl₂-6H₂O, 100 mg/L casein hydrolysate, 0.69 g/L L-proline, 20 g/Lsucrose, 2 g/L GELGRO™, pH 5.8) for five to seven days in the dark at27° C. Somatic embryos mature and shoot regeneration began on medium217. Tissue was transferred to medium 127T (MS salts, 0.65 mg/L niacin,0.125 mg/L pyridoxine-HCl, 0.125 mg/L thiamine-HCl, 0.125 mg/L Capantothenate, 150 mg/L L-asparagine, 100 mg/L myo-inositol, 10 g/Lglucose, 20 g/L L-maltose, 100 mg/L paromomycin, 5.5 g PHYTAGAR™, pH5.8) for shoot development. Tissue on medium 127T was cultured in thelight at 400-600 lux at 26° C. Plantlets are transferred to soil,preferable 3 inch pots, about four to 6 weeks after transfer to 127Tmedium when the plantlets are about 3 inches tall and have roots. Plantswere maintained for two weeks in a growth chamber at 26° C., followed bytwo weeks on a mist bench in a greenhouse before transplanting to 5gallon pots for greenhouse growth. Plants were grown in the greenhouseto maturity and reciprocal pollinations were made with the inbred CORNOF GERMPLASM A. Seed was collected from plants and used for furtherbreeding activities.

Example 26 Isolation of Nucleic Acids from Plants

Nucleic acids were isolated from leaf tissue of R0 plants, collected andflash frozen in a 96 well collection box, 0 to 2 weeks after plantletswere transferred to soil. Approximately 100 milligrams of tissue wascollected from each plant and stored at −80° C. until analysis.

DNA and RNA were isolated from a single tissue sample using the QiagenRneasy 96™ kit (Qiagen Inc., Valencia, Calif.) with modifications. Onehundred milligrams of frozen tissue was homogenized in 700 μL Rneasy™RTL buffer (Qiagen Inc., Valencia, Calif.) using a Bead Beater™ (BiospecProducts, Bartlesville, Okla.). Samples were centrifuged at 3200 rpm for15 minutes and all of the supernatant transferred the wells of a PromegaWIZARD™ clearing plate (Promega Corporation, Madison, Wis.). The samplesolutions were clarified by vacuum filtration through the clearingplate. The cleared supernatant was used for nucleic acid extractions.

For DNA extractions, 70 μL of the cleared sample was transferred to av-well PCR plate, covered with adhesive foil, and heated to 95° C. for 8minutes. The samples were incubated at 0° C. for five minutes, followedby centrifugation for 3 minutes to remove insoluble materials. ASephadex G-50 gel filtration box (Edge Biosystems, Gaithersburg, Mo.)was conditioned for 2 min at 2000 rpm. Forty μL of the heat-treatedsupernatant was loaded into each well and the box centrifuged for twominutes at 2500 rpm. An additional 20 μL of TE buffer was added to thecolumn effluent and the sample plate was stored at −20° C. untilanalysis.

For RNA extractions, five hundred microliters of cleared solution wastransfer to a clean 96 well sample box. Two hundred and fiftymicroliters of 100% ethanol was added to each sample and the sample wasthoroughly mixed. All of the approximately seven hundred and fiftymicroliters of solution was then loaded into the wells of a QiagenRneasy™ binding plate in a Promega WIZARD™ filtration unit. Five hundredmicroliters of RW1 buffer (Qiagen Inc., Valencia, Calif.) was added toeach well and the buffer removed by vacuum filtration. Eightymicroliters of RNAase free DNAase (Qiagen Inc., Valencia, Calif.) wasadded to each well, incubated at room temperature for 15 minutes theDNAase solution drawn through the wells by vacuum filtration. Anadditional five hundred microliters RW1 buffer (Qiagen Inc., Valencia,Calif.) was added to the wells and the buffer removed by vacuumfiltration. The sample was further washed by vacuum filtration with 500μL RPE buffer 2× (Qiagen, Valencia, Calif.). The extraction plate wasplaced on a microtiter plate and centrifuged for three minutes at 3000rpm to remove any residual RPE buffer solution in the filter. Eightymicroliters of RNA grade water (DNAse free) was added to each well,followed by incubation at room temperature for two minutes. Theextraction plate and microtiter plate were centrifuged for three minutesat 3000 rpm and the RNA preparation stored frozen in the collectionplate at −80° C.

Example 27 Assays for Copy Number

Copy number of transgenes in R0 plants was determined using TAQMAN®methods. The pMON65154 and pRG76 GATEWAY™ destination vectors wereconstructed with a sequence derived from the 3′ region of the potatopinII gene which could be used to assay copy number of transgeneinsertions. The pinII forward and reverse primers were as follows:

Forward primer (SEQ ID NO: 77) 5′ ccccaccctgcaatgtga 3′ Reverse primer(SEQ ID NO: 78) 5′ tgtgcatccttttatttcatacattaattaa 3′

The pinII TAQMAN® probe sequence was

(SEQ ID NO: 79) 5′ cctagacttgtccatcttctggattggcca 3′

The probe was labelled at the 5′ end with the fluorescent dye FAM(6-carboyxfluorescein) and the quencher dye TAMRA(6-carboxy-N,N,N′,N′-tetramethylrhodamine) was attached via a linker tothe 3′ end of the probe. The TAQMAN® probe was obtained from AppliedBiosystems (Foster City, Calif.). SAT, a single copy maize gene was usedas an internal control in TAQMAN® copy number assays. The SAT primerswere as follows

Forward primer (SEQ ID NO: 80) 5′ gcctgccgcagaccaa 3′ Reverse primer(SEQ ID NO: 81) 5′ atgcagagctcagettcatc 3′

The SAT TAQMAN® probe sequence was

(SEQ ID NO: 82) 5′ tccagtacgtgcagtccctcctcc 3′

the probe was labelled at its 5′ end with the fluorescent dye VIC™(Applied Biosystems, Foster City, Calif.) and the quencher dye TAMRA atis 3′end.

TAQMAN® PCR was performed according to the manufacturer's instructions(Applied Biosystems, Foster City, Calif.). Five to 100 nanograms DNA wasused in each assay. PCR amplification and TAQMAN® probe detection wereperformed in 1× TAQMAN® Universal PCR Master Mix (Applied Biosystems,Foster City, Calif.) which contains AmpliTaq Gold® DNA polymerase,AmpErase® UNG, dNTPs with dUTP, Passive Reference 1, and optimizedbuffer. Eight hundred nM each forward and reverse pinII primers and 150nM pinII TAQMAN® probe were used in the TAQMAN® assay. 200 nM each Satforward and reverse primers and 150 nM Sat TAQMAN® probe were used inthe TAQMAN® copy number assay. TAQMAN® PCR was carried out for 2 minutesat 50° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at95° C. and one minute at 60° C. Real time TAQMAN® probe fluorescence wasmeasured using an ABI Prism 7700 Sequence Detection System or ABI7900HTSequence Detection System (Applied Biosystems, Foster City, Calif.).C_(T) values were calculated according to the TAQMAN®EZ RT-PCR kitinstruction manual (Applied Biosystems, Foster City, Calif.). TheΔΔC_(T) value was calculated as C_(T) (internal control gene(Sat))−C_(T) (transgene)−C_(T) (internal control gene (Sat) innontransgenic plant). The copy number was assigned as follows accordingto the criteria in Table 19.

TABLE 19 Critera for Copy Number Determination by TAQMAN ® Copy NumberCriteria 1 −0.5 < ^(ΔΔC) _(T) < 0.50 2   0.5 < ^(ΔΔC) _(T) < 1.50 >2^(ΔΔC) _(T) > 1.50

Plants comprising genes of the present invention will be analyzed byTAQMAN® methods for copy number. Southern blot analysis to confirm theTAQMAN® copy number determination in about 80% of the plants that wereanalyzed by both TAQMAN® and Southern blot hybridization.

Example 28 Assays for Gene Expression

Expression of a transgene of the present invention was assayed byTAQMAN® RT-PCR using the TAQMAN® EZ RT-PCR kit from Applied Biosystems(Foster City, Calif.). RNA expression was assayed relative to expressionin a transgenic standard, a transgenic maize event designated DBT418,comprising a B. thuringiensis cryIAI gene operably linked to a pinII 3′untranslated region. The DBT418 event expresses the cryIAI gene at alevel which confers commercial levels of resistance to lepdiopteraninsects such as European Corn Borer and was commercially sold by DEKALBGenetics Corporation under the brand name DEKALBt®. The pMON65154 andpRG76 GATEWAY™ destination vectors were constructed with a sequencederived from the 3′ region of the potato pinII gene which could be usedto assay transgene transcript levels for any coding sequence insertedinto the Destination Vector. The pinII primers and probe previouslydescribed in were used for TAQMAN® RT-PCR. Ubiquitin fusion protein(UBI) RNA was used as an internal control in all TAQMAN® RT-PCR assays.The UBI primers used were as follows:

Forward primer (SEQ ID NO: 83) 5′ cgtctacaatcagaaggcgtaatc 3′ Reverseprimer (SEQ ID NO: 84) 5′ ccaacaggtgaatgcttgatagg 3′

The sequence of the UBI TAQMAN® probe was

(SEQ ID NO: 85) 5′ catgcgccgctttgcttc 3′

The UBI TAQMAN® probe was labeled at its 5′ end with the fluorescent dyeVIC™ (Applied Biosystems, Foster City, Calif.) and the quencher dyeTAMRA at is 3′ end

Reverse transcription, PCR amplification and TAQMAN® probing wereperformed according to the one step procedure described in the TAQMAN®EZ RT-PCR kit (Applied Biosystems, Foster City, Calif.). Five to 100nanograms total RNA was used in each assay. In vitro transcribed controlRNA from the DBT418 event was included as a control on every plate andrun over a concentration range from 0.01 picograms to 10 picograms.Total RNA from DBT418 leaf and from the non-transgenic inbred CORN OFGERMPLASM A were run as positive and negative controls respectively.RT-PCR was performed in TAQMAN® EZ Buffer (50 mM Bicine, 115 mMpotassium acetate, 0.01 mM EDTA, 60 mM Passive Reference 1, 8% glycerol,pH 8.2, Applied Biosystems, Foster City, Calif.) containing 3 mMmanganese acetate, 300 μM each dATP, dCTP, dGTP, and dUTP, 100 unitsrTth™ (Applied Biosystems, Foster City, Calif.) DNA polymerase, and 25units AmpErase UNG (Applied Biosytems, Foster City, Calif.). RT-PCR wascaned out as follows: 2 minutes at 50° C., 30 minutes at 60° C., 5minutes at 95° C., followed by 40 cycles of 20 seconds at 95° C. and 1minute at 60° C. 400 nM each forward and reverse primers were used foramplification of the pinII sequence and 200 nM TAQMAN® pinII probe usedfor detection. UBI RNA was amplified using 400 nM each forward andreverse primers and 200 nM UBI TAQMAN® probe was used for detection.TAQMAN® fluorescence was measured using an ABI Prism 7700 SequenceDetection System or ABI7900HT Sequence Detection System (AppliedBiosystems, Foster City, Calif.). Expression of transgenes of thepresent invention was quantitated relative to transgene expression inDBT418 and reported as a ratio of transgene expression to DBT418expression, i.e., 2^(−(ΔΔC) ^(T) ⁾ (transgene)/2^(−(ΔΔC) ^(T) ⁾(DBT418).

Example 29 Plant Breeding

Backcrossing can be used to improve a starting plant. Backcrossingtransfers a specific desirable trait from one source to an inbred orother plant that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriategene(s) for the trait in question, for example, a construct prepared inaccordance with the current invention. The progeny of this cross firstare selected in the resultant progeny for the desired trait to betransferred from the non-recurrent parent, then the selected progeny aremated back to the superior recurrent parent (A). After five or morebackcross generations with selection for the desired trait, the progenyare hemizygous for loci controlling the characteristic beingtransferred, but are like the superior parent for most or almost allother genes. The last backcross generation would be selfed to giveprogeny which are pure breeding for the gene(s) being transferred, i.e.one or more transformation events.

Therefore, through a series of a breeding manipulations, a selectedtransgene may be moved from one line into an entirely different linewithout the need for further recombinant manipulation. Transgenes arevaluable in that they typically behave genetically as any other gene andcan be manipulated by breeding techniques in a manner identical to anyother corn gene. Therefore, one may produce inbred plants which are truebreeding for one or more transgenes. By crossing different inbredplants, one may produce a large number of different hybrids withdifferent combinations of transgenes. In this way, plants may beproduced which have the desirable agronomic properties frequentlyassociated with hybrids (“hybrid vigor”), as well as the desirablecharacteristics imparted by one or more transgene(s).

It is desirable to introgress the genes of the present invention intomaize hybrids for characterization of the phenotype conferred by eachgene in a transformed plant. The host genotype into which the transgenewas introduced, preferably CORN OF GERMPLASM A, is an elite inbred andtherefore only limited breeding is necessary in order to produce highyielding maize hybrids. The transformed plant, regenerated from callusis crossed, to the same genotype, e.g., CORN OF GERMPLASM A. The progenyare self pollinated twice and plants homozygous for the transgene areidentified. Homozygous transgenic plants are crossed to a testcrossparent in order to produce hybrids. The test cross parent is an inbredbelonging to a heterotic group which is different from that of thetransgenic parent and for which it is known that high yielding hybridscan be generated, for example hybrids are produced from crosses of CORNOF GERMPLASM A to either CORN OF GERMPLASM E or CORN OF GERMPLASM B.

Example 30 Methods of Evaluating Phenotype

Expression of the genes of the present invention leads to variousphenotypes as disclosed herein in transformed cells and plants.Phenotypic data is collected during the transformation process in callusas well as during plant regeneration, as well as in plants and progeny.Phenotypic data is collected in transformed callus relating to themorphological appearance as well as growth of the callus, e.g., shooty,rooty, starchy, mucoid, non-embryogenic, increased growth rate,decreased growth rate, dead. It is expected that one of skill in the artmay recognize other phenotypic characteristics in transformed callus.

Phenotypic data is also collected during the process of plantregeneration as well as in regenerated plants transferred to soil.Phentoypic data includes characteristics such as normal plants, bushyplants, narrow leaves, striped leaves, knotted phenotype, chlorosis,albino, anthocyanin production, buggy whipped (a phenomenon known to theart in which the most recently emerged leaves are elongated and wraparound each other), or altered tassels, ears or roots. It is expectedthat one of skill in the art may recognize other phenotypiccharacteristics in transformed plants.

A wide variety of phenotypes are monitored during the process of plantbreeding and testing in both inbred and hybrid plants. For example, inR0 and R1 plants (plants directly regenerated from callus and the directprogeny of those plants), plant type (general morphologicalcharacteristics such as those described above for plantlets) andnutritional composition of grain produced by the plants are recorded.Nutritional composition analysis may include amino acid composition,amount of protein, starch and oil, characteristics of protein, starchand oil, fiber, ash, mineral content may all be measured. It is expectedthat one of skill in the art may include analyses of other components ofthe grain. In R2 and R3 plants, days to pollen shed, days to silking,and plant type are observed. Furthermore, metabolite profiling of R2plants is conducted. Using methods available to those of skill in theart, 50 to 100 or more metabolites may be analyzed in a plant, therebyestablishing a metabolic fingerprint of the plant. In addition in R3plants, leaf extension rate is measured under field conditions. Avariety of phenotypes will be assayed in hybrids comprising a transgeneof the present invention. For example, yield, moisture, test weight,nutritional composition, chlorophyll content, leaf temperature, stand,seedling vigor, plant height, leaf number, tillering, brace roots, staygreen, stalk lodging, root lodging, plant health, barreness/prolificacy,green snap, pest resistance (including diseases, viruses and insects)and metabolic profiles will be recorded. In addition, phenotypiccharacteristics of grain harvested from hybrids will be recorded,including number of kernels per row on the ear, number of rows ofkernels on the ear, kernel abortion, kernel weight, kernel size, kerneldensity and physical grain quality. Furthermore, characteristics such asphotosynthesis, leaf area, husk structure, kernel dry down rate andinternode length may be measured in hybrids or inbreds. It is expectedthat transcriptional profiling may be performed on transgenic plantsexpressing genes of the present invention.

In order to determine hybrid yield in transgenic plants expressing genesof the present invention, it is recognized that hybrids must be testedat multiple locations in a geographical location where maize isconventionally grown, e.g., Iowa, Illinois or other locations in themidwestern United States. It is expected that more than one year ofyield testing is desirable in order to identify transgenes whichcontribute to improvement of a maize hybrid. Therefore, transgenichybrids will be evaluated in a first year at a sufficient number oflocations to identify at least an approximately 10% yield differencefrom a non-transgenic hybrid counterpart. A second year of yield testsis conducted at sufficient locations and with sufficient repetitions tobe able to identify a 4-5% yield difference between two hybrids.Furthermore, in the second year of yield tests, hybrids will beevaluated under normal field conditions as well as under stressconditions, e.g., under conditions of water or population densitystress. One of skill in the art knows how to design a yield trial suchthat a statistically significant yield difference can be detectedbetween two hybrids at the desired rate of precision.

Example 31 Surface Sterilization and Imbibition of Corn Seeds

For each transgenic lot, surface sterilize about 50 corn seeds byputting them in a sterile 500 ml Erlenmyer flask with 50 ml of 30%bleach (sodium hypochlorite solution=Chlorox or equivalent) solutioncontaining 0.01% triton X-100 and rotating the flask on an orbitalshaker for 5 minutes. Then pour off the bleach solution and wash withabout 100 ml of sterile deionized water and pour off the water wash.Repeat the sterile water wash 4 more times, leaving the last water washon the seeds. Incubate the seeds in this water at room temperature for24 h for imbibition under air bubbling (pass through 0.2 μm filter).

I. Preparation of Media in Phytotrays.

Prepare water-agar media for several Phytotrays. We are using PhytotrayII (or plastic box: 60×30×15 cm) in the inverted position so that thelarger depth side of the vessel is on the bottom and the smaller side isused as the lid. Prepare enough water-agar media for 100 ml perPhytotray by autoclaving 0.3% BactoAgar in deionized water for 45minutes on the liquid cycle. Cool the media to the extent it can behandled easily and pour approximately 100 ml per Phytotray while stillmolten.

II. Corn Cold Seedling Vigor Assay.

-   -   When the media has solidified, bring it and the sterile seeds to        a laminar flow hood.    -   Using sterile forceps, select 20 healthy, most uniform seeds and        place the seeds in each Phytotray used for the assay, spacing        the seeds evenly so that any individuals can be easily removed        later.    -   Place seeds so that the embryo side is diagonally inserted        downward and the seed is just under the surface of the agar. In        this position, the emerging shoot and root will be able to        directly elongate without cramping.    -   Incubate the seeds in the media at 22° C. for one week, or until        most of the seeds have extruded radicles and are beginning to        emerge from the agar.    -   Remove all but the 10 most uniformly grown seedlings in a        laminar flow hood.    -   Shift the Phytotrays to a cold plant growth chamber set at        10° C. with 16 hour day cycle and incubate there for 2 weeks.    -   Shift the Phytotrays back to 22° C. for one week.    -   Remove seedlings, measure root length and shoot length for every        seedling, and measure fresh weight g/3 seedlings record in        notebook.

Adaptation for Cold Germination and Emergence Assay.

Same as above with the following exceptions:

-   -   After the last water wash in I., place the flasks at 10° C.        during the overnight imbibition step. Also prechill the        Phytotrays with solidified media at 10° C.    -   After seeding the chilled Phytotrays with cold imbibed seeds,        they are put directly into the 10° C. chamber.    -   After about 5 days, remove all but the 10 most uniformly        germinated seeds, those whose radicles are about the same        length. Return Phytotrays to 10° C. chamber for 1-2 weeks.        Remove seedlings, measure root length and shoot length for every        seedling, and measure fresh weight from every 3 seedlings,        record in notebook.    -   Shift the 2^(nd) set Phytotrays to 22° C. for 1 week.

Remove seedlings, measure root length and shoot length for everyseedling, record in notebook.

Example 32 Creation of Plasmids for Transformation of Soybean Example(for CspA and B Constructs—pMON73983 and 73984)

pMON73983 (FIG. 18) is a binary vector for Agrobacterium-mediatedtransformation and constitutive expression of a protein (SEQ ID NO: 1)like Bacillus subtilis CspA in Soybean. To clone the B. subtilis CspAgene, two gene-specific primers, MSA452 and MSA453 were designed basedon the CspA sequence information (Genbank # M30139) from the NationalCenter for Biotechnology Information, which is part of the NationalLibrary of Medicine, in turn part of the National Institutes of Health(NCBI). The sequence for MSA452 isGCGCAGGCCTAGATGTACCATGTCCGGTAAAATGACTGGTATCGTAAAATGG (SEQ ID NO: 86),which anneals at the translational start site of CspA and introducesStuI and BglII sites at the 5′ end, while the sequence of MSA453 isCGCGAATTCGGATCCTTATTACAGGCTGGTTACGTTACCAGCTGCC (SEQ ID NO: 87), whichanneals at the last codon of CspA and introduces BamHI and EcoRI sitesat the end of the primer. The reverse primer MSA453 was designed tomatch the 3′ end of the Genbank gene sequence. The PCR reaction wascarried out using primers MSA452 and MSA453, High Fidelity TaqPolymerase (BRL) and pMON57397 (FIG. 3) as the template. This templatediffers at the 3′end of the gene CspA, from that of the GeneBanksequence. The amplified CspB DNA was purified by gel-electrophoresis andligated to pCR-XL-TOPO vector (Invitrogen). The ligation reaction wastransformed into E. coli Top10 cells (Invitrogen) as per manufacturer'sprotocol. Four transformant colonies were picked and miniprep DNA wasprepared using Qiagen Miniprep Kit. The inserts were sequenced usingM13-specific Forward and Reverse primers. Clone with the correctsequence was named pMON73981 and used for further subcloning.

PMON73881 DNA was digested with StuI and BamHI to isolate the CspA genefragment. pMON73980 DNA was digested with StuI and BamHI sequentially,and then purified by Gene Clean II kit. The CspB fragment and thispurified vector pMON73980 were ligated and the ligation reaction waselectrotransformed into E. coli DH10 B cells. The transformants wereselected on Spectinomycin containing media. The miniprep DNA wasprepared from the transformants and the DNA was checked for the presenceof the insert by using CaMV35S-promoter-specific forward primer. Theclone containing this insert was named as pMON73983. A larger DNA prepwas made and a series of confirmatory digests were carried out,including BglII, EcoRI, PstI, EcoRI+BamHI, StuI+XhoI. These confirmedthe correct cloning.

pMON73984 is a binary vector for Agrobacterium-mediated transformationand constitutive expression of a protein (SEQ ID NO: 2) like Bacillussubtilis CspB in Arabidopsis. To clone the B. subtilis CspB gene, twogene-specific primers, MSA454 and MSA455 were designed based on the CspBsequence information (Genbank # X59715) from the National Center forBiotechnology Information, which is part of the National Library ofMedicine, in turn part of the National Institutes of Health (NCBI). Thesequence for MSA454 isGCGCAGGCCTAGATGTACCATGTTAGAAGGTAAAGTAAAATGGTTCAACTCTG (SEQ ID NO: 88),which anneals at the translational start site of CspB and introducesStuI and BglII sites at the 5′ end, while the sequence of MSA455 isCGCGAATTCGGATCCTTATTACGCTTCTTTAGTAACGTTAGCAGCTTGTGG (SEQ ID NO: 89),which anneals at the last codon of CspB and introduces BamHI and EcoRIsites at the end of the primer. The reverse primer MSA455 was designedto match the 3′ end of the Genbank gene sequence. The PCR reaction wascarried out using primers MSA454 and MSA455, High Fedelity TaqPolymerase (BRL) and pMON57399 as the template. This template differs atthe 3′end of the gene CspB, from that of the GeneBank sequence. Theamplified CspB DNA was purified by gel-electrophoresis and ligated topCR-XL-TOPO vector (Invitrogen). The ligation reaction was transformedinto E. coli Top10 cells (Invitrogen) as per manufacturer's protocol.Four transformant colonies were picked and miniprep DNA was preparedusing Qiagen Miniprep Kit. The inserts were sequenced using M13-specificForward and Reverse primers. Clone with the correct sequence was namedpMON73982 and used for further subcloning.

PMON73882 DNA was digested with StuI and BamHI to isolate the CspB genefragment. pMON73980 DNA was digested with StuI and BamHI sequentially,and then purified by Gene Clean II kit. The CspB fragment and thispurified vector pMON73980 were ligated and the ligation reaction waselectrotransformed into E. coli DH10 B cells. The transformants wereselected on Spectinomycin containing media. The miniprep DNA wasprepared from the transformants and the DNA was checked for the presenceof the insert by using CaMV35S-promoter-specific forward primer. Theclone containing this insert was named as pMON73984. A larger DNA prepwas made and a series of confirmatory digests were carried out,including BglII, EcoRI, PstI, EcoRI+BamHI, StuI+XhoI. These confirmedthat the cloning was correct.

Soybean plants were created, through transformation, with the pMONconstructs above stably integrated in their genome.

Example 33

Corn plant transformed with DNA constructs from examples 10 and 11,above, were studied.

Greenhouse

-   -   Two experiments were performed, one testing 10 cspA events and        one testing 10 cspB events for drought tolerance.    -   24 transgene positive and 24 transgene negative hybrid seedlings        from each event were tested (all seeds derived from segregating        hybrid ears).    -   The test was performed on benches in a greenhouse.    -   The treatment consisted of withholding water and monitoring        total pot weight of each pot containing a plant. Fully watered        pots weigh about 1000 grams each and water was withheld until        each pot's weight reached 400 grams, then pots were maintained        at that weight during the remainder of the treatment.    -   Throughout the treatment, plant height was determined by        measuring the distance from the soil surface in the pot to the        tip of the “tallest” leaf. From these measurements LER (leaf        extension rates) were determined by comparing the heights at the        intervals between measurements.    -   LER comparisons during the drought were made between transgene        negative and transgene positive plants within an event.    -   For three of ten events tested, cspA transgenic plants were        significantly (p<0.10) improved for LER during the treatment.    -   For three of ten events tested, cspB transgenic plants were        significantly (p<0.10) improved for LER during the treatment.        Field Efficacy    -   Three experiments were performed using hybrid seed, one testing        16 cspB events (CA), one testing 21 cspB events (KS), and one        testing 14 cspA events (HI) for drought tolerance during the        late vegetative stage of growth.    -   For the CA and HI trials, rows containing ˜34 plants,        segregating for presence of the transgene, were present in six        and four replicates, respectively. Segregating rows were derived        from segregating ears.    -   For KS experimental rows contained ˜34 plants; as transgenic and        non-transgenic paired rows, with six replicates.    -   The treatment consisted of withholding water for approximately        ten days during the late vegetative phase of growth (giving a        small amount as needed to maintain viable plants). At the end of        the ten-day period plants were then well irrigated until        harvest.    -   Throughout the treatment a number of phenotypes were measured        including LER, chlorophyll (by SPAD meter), and photosynthesis        rate. Following the treatment additional phenotypes measured        included: days to pollen shed and silk emergence, and ear        components such as kernels/ear, ears with kernels, kernel        weight, and yield.    -   Phenotype comparisons were made between transgene positive and        negative plants within an event and across the construct.    -   In the CA trial, cspB as a construct (across all events for        vegetative traits and across the “best” six events for        reproductive traits) transgene positive plants were        significantly (p<0.10) improved for LER, leaf temperature, and        kernels/ear during or following the drought treatment.    -   In the CA trial, individual events were significantly (p<0.10)        improved for LER, average ear length, kernel mass/ear, stomatal        conductance, and days to silking during or following the drought        treatment.    -   In the KS trial, cspB as a construct (across all events for        vegetative traits and across the “best” six events for        reproductive traits) transgene positive plants were        significantly (p<0.10) improved for LER, kernel bearing        ears/row, kernels/ear, kernels/plant, shell weight, and yield.    -   In the KS trial, individual events were significantly (p<0.10)        improved for LER, photosynthetic rate, stomatal conductance,        ears/row, and kernels/plant.    -   In the HI trial, three events were significantly (p<0.10)        improved for LER (chlorophyll content was the only other        phenotype measured in HI)

Summaries of CA and KS Results:

Summary of Field Efficacy Results for cspB-KS Site

1. The field design, site uniformity, and execution of planting andsampling were all consistent with a high quality experiment capable ofgenerating informative data sets.

2. The water-limited treatment was applied in a manner that resulted intreatment impacts on all phenotypes measured, particularly LER,chlorophyll, and photosynthetic rates.

3. The treatment impacts on vegetative and reproductive phenotypes weresufficient to be statistically real and to allow for transgene-mediatedimprovements to be observed at statistically significant levels.

4. One or more events were statistically improved in transgenecontaining plants for LER, chlorophyll, photosynthetic rate, stomatalconductance leaf temperature, days to pollen shed, days to silking,anthesis silking interval, ears/plot, kernels/ear, kernels/plant, shellweight, and estimated yield.5. Construct level statistical improvement was observed at p<0.10 in thedry treatment for LER, ears/plot, kernels/ear, kernels/plant, shellweight, and estimated yield, and for LER in the wet treatment.

TABLE 20 Event Treatment Improved phenotype P value Construct Dry LER(T1-T0) 0.009 Dry LER (T2-T0) 0.009 Dry LER (T2-T1) 0.096 Dry Stomatalconductance 0.150 Dry Photosynthesis 0.141 Dry Ears/plot 0.012 DryKernels/ear 0.062 Dry Kernels/plant 0.006 Dry Shell weight 0.009 DryEst. Yield 0.008 Wet LER (T2-T1) 0.025 Wet Chlorophyll (—) 0.062 WetEars/plot (—) 0.185 Wet Kernels/ear (—) 0.121 Wet Kernels/plant (—)0.083 Wet Shell weight (—) 0.132 Wet Est. Yield (—) 0.101 ZM_M38835 DryLER (T1-T0) 0.008 Dry Photosynthesis 0.066 Dry Stomatal conductance0.064 Dry Transpiration 0.126 Dry Kernels/plant 0.160 Dry Shell weight0.149 Dry Yield 0.153 Wet LER (T1-T0) 0.099 Wet LER (T2-T1) (—) 0.026ZM_M38737 Dry Photosynthesis 0.108Summary of Field Efficacy Results for cspB-CS Site (Font Change)1. The field design, site uniformity, and execution of planting andsampling were all consistent with a high quality experiment capable ofgenerating informative data sets.2. The water-limited treatment was applied in a manner that resulted intreatment impacts on all vegetative phenotypes measured, particularlyLER, chlorophyll, and photosynthetic rates, but not on all reproductivephenotypes.3. The treatment impacts on phenotypes (vegetative) of interest weresufficient to be statistically real and to allow for transgene-mediatedimprovements to be observed at statistically significant levels.4. One or more events were statistically improved in transgenecontaining plants for LER, chlorophyll, photosynthetic rate, stomatalconductance leaf temperature, days to pollen shed, days to silking,anthesis silking interval, kernels/ear, average ear length, and kernelmass/ear.5. Construct level statistical improvement was observed in the drytreatment for LER, leaf temperature, and days to pollen shed, and forASI in the wet treatment.

TABLE 21 Event Treatment Improved phenotype P value Construct Dry LER0.009 Dry Leaf temperature 0.027 Dry Days to pollen shed 0.192 DryKernels/ear 0.080 Dry Kernel mass/ear 0.197 Dry Test Wt (lb/bu) Neg0.084 Wet LER 0.157 Wet Days to pollen shed 0.098 Wet Ave ear length0.091 Wet Kernel mass/ear 0.010 Wet Test Wt (lb/bu) Neg 0.188 ZM_M39583Dry LER 0.051 Dry Kernels/ear 0.200 Wet Ave ear length 0.058 Wet Kernelmass/ear 0.070 ZM_M39872 Dry LER 0.159 Wet Days to silking 0.024 Wet ASI0.064 ZM_M40946 Dry LER 0.201 ZM_M38238 Dry Days to silking 0.176 DryKernels/ear 0.192 Wet LER 0.151 Wet Kernel mass/ear 0.034 ZM_M38244 DryStomatal Conductance 0.092 Dry Photosynthesis 0.132 Dry Leaf Temperature0.155 ZM_M38230 Dry Days to silking 0.176 ZM_M38721 Dry Days to silking0.066 Dry ASI 0.109 Wet Days to silking 0.117 ZM_M38714 Wet Days tosilking 0.010 Wet ASI 0.025 ZM_M40939 Dry ASI 0.109Many of these events have been subsequently tested for improvements incold germination efficiency and seedling growth under cold conditions,and have not proved efficacious. Thus, these genes driven by thispromoter are unlikely to function in maize for improvement of coldgermination or seedling growth under cold conditions, but differentpromoters driving the same genes, or different cold shock proteins mayfunction in maize to improve these phenotypes.

What is claimed is:
 1. A drought tolerant transgenic plant thatcomprises in its genome a recombinant DNA molecule that expresses a coldshock protein, wherein said cold shock protein comprises the cold shockdomain sequence [FY]-G-F-I-x(6,7)-[DER]-[LIVM]-F-x-H-x-[STKR]-x-[LIVMFY]of SEQ ID NO:3, has at least 60% identity across the entire length ofthe Escherichia coli CspA protein of SEQ ID NO:1, and confers resistanceto drought.
 2. The drought tolerant transgenic plant of claim 1, whereinsaid cold shock protein has at least 70% identity across the entirelength of Escherichia coli CspA protein of SEQ ID NO:1.
 3. The droughttolerant transgenic plant of claim 1, wherein said cold shock proteinhas at least 80% identity across the entire length of Escherichia coliCspA protein of SEQ ID NO:1.
 4. The drought tolerant transgenic plant ofclaim 1, wherein said cold shock protein has at least 90% identityacross the entire length of Escherichia coli CspA protein of SEQ IDNO:1.
 5. The drought tolerant transgenic plant of claim 1, wherein saidcold shock protein has at least 95% identity across the entire length ofEscherichia coli CspA protein of SEQ ID NO:1.
 6. The drought toleranttransgenic plant of claim 1, wherein said transgenic plant is a monocotor a dicot plant.
 7. The drought tolerant transgenic plant of claim 1,wherein said transgenic plant is a soybean, corn, canola, rice, cotton,barley, oat, turf grass, alfalfa, or wheat plant.
 8. The transgenicplant of claim 1, wherein said plant has an increased yield whencompared to a non-transformed plant of the same species when saidtransgenic plant and said non-transformed plant are grown under droughtstress.
 9. A transgenic propagule of the drought tolerant transgenicplant of claim 1, wherein said propagule comprises in its genome arecombinant DNA molecule that expresses a cold shock protein, whereinsaid cold shock protein comprises the cold shock domain sequence[FY]-G-F-I-x(6,7)-[DER]-[LIVM]-F-x-H-x-[STKR]-x-[LIVMFY] of SEQ ID NO:3,has at least 60% identity across the entire length of the Escherichiacoli CspA protein of SEQ ID NO:1 and confers drought resistance to atransgenic plant comprising said recombinant DNA that is produced fromsaid propagule.
 10. The transgenic propagule of claim 9, wherein saidcold shock protein has at least 80% identity across the entire length ofEscherichia coli CspA protein of SEQ ID NO:1.
 11. The transgenicpropagule of claim 9, wherein said cold shock protein has at least 90%identity across the entire length of Escherichia coli CspA protein ofSEQ ID NO:1.
 12. The transgenic propagule of claim 9, wherein saidpropagule is a seed.
 13. The transgenic propagule of claim 9, whereinsaid propagule is a root, shoot, leaf, stem, embryo, or cell.
 14. Amethod of improving drought tolerance in a transgenic progeny plantcomprising crossing a plant with a drought tolerant transgenic planthaving a recombinant DNA expressing a cold shock protein wherein saidcold shock protein comprises a cold shock domain sequence[FY]-G-F-I-x(6,7)-[DER]-[LIVM]-F-x-H-x-[STKR]-x-[LIVMFY] of SEQ ID NO:3,has at least 60% identity across the entire length of the Escherichiacoli CspA protein of SEQ ID NO:1, and confers resistance to drought, andobtaining a transgenic progeny plant having said recombinant DNA. 15.The method of claim 14, wherein said transgenic plant is a soybean,corn, canola, rice, cotton, barley, oat, turf grass, alfalfa, or wheatplant.
 16. A method of producing a drought tolerant transgenic plantcomprising the steps of: a) inserting into the genome of plant cells arecombinant DNA molecule that comprises, in the 5′ to 3′ direction: (i)a first DNA polynucleotide comprising a promoter that functions inplants and which is operably linked to (ii) a second DNA polynucleotidethat encodes a cold shock protein that comprises a cold shock domainsequence [FY]-G-F-I-x(6,7)-[DER]-[LIVM]-F-x-H-x-[STKR]-x-[LIVMFY] of SEQID NO:3, has at least 60% identity across the entire length ofEscherichia coli CspA protein of SEQ ID NO:1, and which is operablylinked to (iii) a 3′ transcription termination DNA polynucleotide thatfunctions as a polyadenylation sequence; b) obtaining transformed plantcells containing said recombinant DNA; c) regenerating transgenic plantsfrom said plant cells; and d) selecting a transgenic plant havingincreased drought tolerance.
 17. The method of claim 16, wherein saidcold shock protein has at least 70% identity across the entire length ofEscherichia coli CspA protein of SEQ ID NO:1.
 18. The method of claim16, wherein said cold shock protein has at least 80% identity across theentire length of Escherichia coli CspA protein of SEQ ID NO:1.
 19. Themethod of claim 16, wherein said cold shock protein has at least 90%identity across the entire length of Escherichia coli CspA protein ofSEQ ID NO:1.
 20. The method of claim 16, wherein said cold shock proteinhas at least 95% identity across the entire length of Escherichia coliCspA protein of SEQ ID NO:1.
 21. The method of claim 16, wherein saidtransgenic plant is a monocot or a dicot plant.
 22. The method of claim16, wherein said transgenic plant is a soybean, corn, canola, rice,cotton, barley, oat, turf grass, alfalfa, or wheat plant.
 23. The methodof claim 16, wherein said transgenic plant has an increased yield whencompared to a non-transformed plant of the same species and when saidtransgenic plant and said non-transformed plant are grown under droughtstress.
 24. A method for increasing yield in a crop subject to waterdeficit during its growth, said method comprising planting seeds havinga recombinant DNA expressing a cold shock protein, wherein said coldshock protein comprises the cold shock domain sequence[FY]-G-F-I-x(6,7)-[DER]-[LIVM]-F-x-H-x-[STKR]-x-[LIVMFY] of SEQ ID NO:3,has at least 60% identity across the entire length of the Escherichiacoli CspA protein of SEQ ID NO:1 and confers resistance to drought, andallowing said seeds to grow to mature plants under drought conditions.25. The method of claim 24, wherein said cold shock protein has at least70% identity across the entire length of Escherichia coli CspA proteinof SEQ ID NO:1.
 26. The of claim 24, wherein said cold shock protein hasat least 80% identity across the entire length of Escherichia coli CspAprotein of SEQ ID NO:1.
 27. The method of claim 24, wherein said coldshock protein has at least 90% identity across the entire length ofEscherichia coli CspA protein of SEQ ID NO:1.
 28. The method of claim24, wherein said cold shock protein has at least 95% identity across theentire length of Escherichia coli CspA protein of SEQ ID NO:1.
 29. Themethod of claim 24, wherein said plants are soybean, corn, canola, rice,cotton, barley, oat, turf grass, alfalfa, or wheat plants.